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Sample records for chitin synthase inhibitor

  1. Synthesis and biological evaluation of novel phosphoramidate derivatives of coumarin as chitin synthase inhibitors and antifungal agents.

    PubMed

    Ji, Qinggang; Ge, Zhiqiang; Ge, Zhixing; Chen, Kaizhi; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Yang, Xiaolan; Liao, Fei

    2016-01-27

    A series of novel phosphoramidate derivatives of coumarin have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their chitin synthase inhibition activity and antimicrobial activity invitro. The bioactive assay manifested that most of the target compounds exhibited good efficacy against CHS and a variety of clinically important fungal pathogens. In particular, compound 7t with IC50 of 0.08mM against CHS displayed stronger efficiency than the reference Polyoxin B with IC50 of 0.16mM. In addition, the apparent Ki values of compound 7t was 0.096mM while the Km of Chitin synthase prepared from Candida tropicalis was 3.86mM for UDP-N-acetylglucosamine, and the result of the Ki showed that the compounds was a non-competitive inhibitor of the CHS. As far as the antifungal activity is concerned, compounds 7o, 7r and 7t were highly active against Aspergillus flavus with MIC values in the range of 1?g/mL to 2?g/Ml while the results of antibacterial screening showed that these compounds have negligible actions to the tested bacteria. These results indicated that the design of these compounds as antifungal agents was rational. PMID:26647304

  2. The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

    PubMed Central

    Schnitzer, Veronika; Weiss, Ingrid M

    2007-01-01

    Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 ?M 10 ?M) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types. PMID:17986326

  3. Benzoylurea Chitin Synthesis Inhibitors.

    PubMed

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs. PMID:26168369

  4. Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents.

    PubMed

    Ge, Zhiqiang; Ji, Qinggang; Chen, Chunyan; Liao, Qin; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Liao, Fei

    2016-04-01

    A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC50 of 0.10 mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC50 of 0.18 mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4 μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents. PMID:25815669

  5. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

    PubMed Central

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a T. urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  6. 2-acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases (CSs) transfer GlcNAc from UDP-GlcNAc to pre-existing chitin chains in reactions that are typically stimulated by free GlcNAc. The eff...

  7. Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs

    PubMed Central

    Guerriero, Gea; Avino, Mariano; Zhou, Qi; Fugelstad, Johanna; Clergeot, Pierre-Henri; Bulone, Vincent

    2010-01-01

    Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control. PMID:20865175

  8. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

    PubMed Central

    Herasimenka, Yury; Kotasinska, Marta; Walter, Stefan; Schrempf, Hildgund

    2010-01-01

    A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes. PMID:20957083

  9. Characterization of a Chitin Synthase Encoding Gene and Effect of Diflubenzuron in Soybean Aphid, Aphis Glycines

    PubMed Central

    Bansal, Raman; Mian, M. A. Rouf; Mittapalli, Omprakash; Michel, Andy P.

    2012-01-01

    Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression. PMID:23139631

  10. Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We have identified and characterized a chitin synthase gene (CHS) from cDNA of Aphis glycines, the soybean aphid, a serious pest of soybean. The full-length cDNA of CHS in A. glyc...

  11. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae.

    PubMed

    Demaeght, Peter; Osborne, Edward J; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans; Clark, Richard M; Van Leeuwen, Thomas

    2014-08-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as 'mite growth inhibitors', and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  12. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    SciTech Connect

    Schoenitzer, Veronika; Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg ; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  13. 2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2*

    PubMed Central

    Gyore, Jacob; Parameswar, Archana R.; Hebbard, Carleigh F. F.; Oh, Younghoon; Bi, Erfei; Demchenko, Alexei V.; Price, Neil P.; Orlean, Peter

    2014-01-01

    Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues. PMID:24619411

  14. Chitin synthases are required for survival, fecundity and egg-hatch in the red flour beetle, Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis of chitin, the Beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, ...

  15. Chitin synthesis inhibitors: old molecules and new developments.

    PubMed

    Merzendorfer, Hans

    2013-04-01

    Abstract  Chitin is the most abundant natural aminopolysaccharide and serves as a structural component of extracellular matrices. It is found in fungal septa, spores, and cell walls, and in arthropod cuticles and peritrophic matrices, squid pens, mollusk shells, nematode egg shells, and some protozoan cyst walls. As prokaryotes, plants and vertebrates including humans do not produce chitin, its synthesis is considered as an attractive target site for fungicides, insecticides, and acaricides. Although no chitin synthesis inhibitor has been developed into a therapeutic drug to treat fungal infections in humans, a larger number of compounds have been successfully launched worldwide to combat arthropod pests in agriculture and forestry. This review summarizes the latest advances on the mode of action of chitin synthesis inhibitors with a special focus on those molecules that act on a postcatalytic step of chitin synthesis. PMID:23955853

  16. Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

    PubMed Central

    Rast, D M; Bartnicki-Garcia, S

    1981-01-01

    The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely. PMID:6453344

  17. Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

    PubMed Central

    Van Leeuwen, Thomas; Demaeght, Peter; Osborne, Edward J.; Dermauw, Wannes; Gohlke, Simon; Nauen, Ralf; Grbić, Miodrag; Tirry, Luc; Merzendorfer, Hans; Clark, Richard M.

    2012-01-01

    Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods. PMID:22393009

  18. Putative chitin synthases from Branchiostoma floridae show extracellular matrix-related domains and mosaic structures.

    PubMed

    Guerriero, Gea

    2012-08-01

    The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans. PMID:23084775

  19. Cell wall protection by the Candida albicans class I chitin synthases

    PubMed Central

    Preechasuth, Kanya; Anderson, Jeffrey C.; Peck, Scott C.; Brown, Alistair J.P.; Gow, Neil A.R.; Lenardon, Megan D.

    2015-01-01

    Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2?chs8? mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses. PMID:26257018

  20. Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger

    SciTech Connect

    Rinker, Torri E.; Baker, Scott E.

    2007-01-29

    Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In this pelleted form, A. niger has increased citric acid production as compared to growth in filamentous form. Chitin is a crucial component of the cell wall of filamentous fungi. Alterations in the deposition or production of chitin may have profound effects on the morphology of the organism. In order to study the role of chitin synthesis in pellet formation we have deleted a chitin synthase gene (csmA) in Aspergillus niger strain ATCC 11414 using a PCR based deletion construct. This class of chitin synthases is only found in filamentous fungi and is not present in yeasts. The csmA genes contain a myosin motor domain at the N-terminus and a chitin synthesis domain at the C-terminus. They are believed to contribute to the specialized polar growth observed in filamentous fungi that is lacking in yeasts. The csmA deletion strain (csmAΔ) was subjected to minimal media with and without osmotic stabilizers as well as tested in citric acid production media. Without osmotic stabilizers, the mutant germlings were abnormally swollen, primarily in the subapical regions, and contained large vacuoles. However, this swelling is ultimately not inhibitory to growth as the germlings are able to recover and undergo polar growth. Colony formation was largely unaffected in the absence of osmotic stabilizers. In citric acid production media csmAΔ was observed to have a 2.5 fold increase in citric acid production. The controlled expression of this class of chitin synthases may be useful for improving production of organic acids in filamentous fungi.

  1. Fusarium verticillioides chitin synthases CHS5 and CHS7 are required for normal growth and pathogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium verticillioides is both an endophyte and a pathogen of maize and is a health threat in many areas of the world because it can contaminate maize with fumonisins, a toxic secondary metabolite. We identified eight putative chitin synthase (CHS) genes in F. verticillioides genomic sequence and...

  2. Construction of novel vectors for transformation of Lentinula edodes using a chitin synthase gene promoter.

    PubMed

    Sato, Toshitsugu; Okawa, Kumiko; Hirano, Tatsuya

    2011-02-01

    Novel vectors (pLCHS-hph and pChG-bar) containing expression unit driving hph as a selectable marker gene by chitin synthase gene promoter were constructed for Lentinula edodes transformation. Expression of the hph gene in random selected transformants was confirmed by RT-PCR method. Thus, both vectors are useful for L. edodes transformation. PMID:21044863

  3. Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy.

    PubMed Central

    Mehmann, B; Brunner, I; Braus, G H

    1994-01-01

    DNA sequences of single-copy genes coding for chitin synthases (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase; EC 2.4.1.16) were used to characterize ectomycorrhizal fungi. Degenerate primers deduced from short, completely conserved amino acid stretches flanking a region of about 200 amino acids of zymogenic chitin synthases allowed the amplification of DNA fragments of several members of this gene family. Different DNA band patterns were obtained from basidiomycetes because of variation in the number and length of amplified fragments. Cloning and sequencing of the most prominent DNA fragments revealed that these differences were due to various introns at conserved positions. The presence of introns in basidiomycetous fungi therefore has a potential use in identification of genera by analyzing PCR-generated DNA fragment patterns. Analyses of the nucleotide sequences of cloned fragments revealed variations in nucleotide sequences from 4 to 45%. By comparison of the deduced amino acid sequences, the majority of the DNA fragments were identified as members of genes for chitin synthase class II. The deduced amino acid sequences from species of the same genus differed only in one amino acid residue, whereas identity between the amino acid sequences of ascomycetous and basidiomycetous fungi within the same taxonomic class was found to be approximately 43 to 66%. Phylogenetic analysis of the amino acid sequence of class II chitin synthase-encoding gene fragments by using parsimony confirmed the current taxonomic groupings. In addition, our data revealed a fourth class of putative zymogenic chitin synthesis. Images PMID:7944356

  4. The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases

    PubMed Central

    Schwelm, Arne; Fogelqvist, Johan; Knaust, Andrea; Jülke, Sabine; Lilja, Tua; Bonilla-Rosso, German; Karlsson, Magnus; Shevchenko, Andrej; Dhandapani, Vignesh; Choi, Su Ryun; Kim, Hong Gi; Park, Ju Young; Lim, Yong Pyo; Ludwig-Müller, Jutta; Dixelius, Christina

    2015-01-01

    Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes. PMID:26084520

  5. The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases.

    PubMed

    Schwelm, Arne; Fogelqvist, Johan; Knaust, Andrea; Jlke, Sabine; Lilja, Tua; Bonilla-Rosso, German; Karlsson, Magnus; Shevchenko, Andrej; Dhandapani, Vignesh; Choi, Su Ryun; Kim, Hong Gi; Park, Ju Young; Lim, Yong Pyo; Ludwig-Mller, Jutta; Dixelius, Christina

    2015-01-01

    Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes. PMID:26084520

  6. Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths.

    PubMed

    Shirk, Paul D; Perera, Omaththage P; Shelby, Kent S; Furlong, Richard B; LoVullo, Eric D; Popham, Holly J R

    2015-12-10

    Chitin is an extracellular biopolymer that contributes to the cuticular structural matrix in arthropods. As a consequence of its rigid structure, the chitinous cuticle must be shed and replaced to accommodate growth. Two chitin synthase genes that encode for chitin synthase A (ChSA), which produces cuticular exoskeleton, and chitin synthase B (ChSB), which produces peritrophic membrane, were characterized in the genomes of two heliothine moths: the corn earworm/cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). In both moths, the two genes were arranged in tandem with the same orientation on the same strand with ChSB located 5' of ChSA. Sequence comparisons showed that the coding sequences were highly conserved with homologues from other species but that the tandem juxtaposed genomic arrangement of the two genes was unique in these insects. The mechanism that has led to this arrangement is unclear but is most likely a recent recombinational event. Transcript mapping of HzChSB and HzChSA in H. zea demonstrated that both transcripts were differentially spliced in various tissues and larval stages. The identification of the HzChSB-E12b alternate spliced transcript is the first report of alternate splicing for the ChSB group. The importance of this splice form is not clear because the protein produced would lack any enzymatic activity but retain the membrane insertion motifs. As for other insects, these genes provide an important target for potential control through RNAi but also provide a subject for broad scale genomic recombinational events. PMID:26253161

  7. Molecular cloning, expression pattern and comparative analysis of chitin synthase gene B in Spodoptera exigua.

    PubMed

    Kumar, N Senthil; Tang, Bin; Chen, Xiaofei; Tian, Honggang; Zhang, Wenqing

    2008-03-01

    The chitin synthase (CHS) gene B (4781 bp) of Spodoptera exigua (SeCHSB) was cloned by reverse-transcription PCR (RT-PCR) and 3'/5' RACE from the midgut. SeCHSB contains an open reading frame of 4572 nucleotides, encoding a protein of 1523 amino acids with a predicted molecular mass of approximately 174.6 kDa. Alignment of SeCHSB with class B CHSs of other insects showed a high degree of conservation in the putative catalytic domain region. The structure of the SeCHSB gene was analyzed and was found to be the same as that of Manduca sexta CHSB (MsCHSB), including 23 exons and 22 introns but without alternative exons. Southern blot analysis revealed that SeCHSB was a single copy gene and the presence of only two chitin synthase genes in S. exigua. Further investigation indicated that SeCHSB was specifically expressed in the midgut, and its transcript existed constitutively in the midgut from the 3rd instar larval stage to prepupae and reached highest expression on the 1st day of the fifth instar larval stage. These data suggest that SeCHSB is very important in midgut formation and development. Chitin synthase gene comparisons between different classes of insects using software tools revealed some interesting aspects of the similarity and divergence of the gene in the Class Insecta. PMID:18178495

  8. Exposure to Diflubenzuron Results in an Up-Regulation of a Chitin Synthase 1 Gene in Citrus Red Mite, Panonychus citri (Acari: Tetranychidae)

    PubMed Central

    Xia, Wen-Kai; Ding, Tian-Bo; Niu, Jin-Zhi; Liao, Chong-Yu; Zhong, Rui; Yang, Wen-Jia; Liu, Bin; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB. PMID:24590130

  9. Hyaluronan synthase assembles chitin oligomers with -GlcNAc(?1?)UDP at the reducing end.

    PubMed

    Weigel, Paul H; West, Christopher M; Zhao, Peng; Wells, Lance; Baggenstoss, Bruce A; Washburn, Jennifer L

    2015-06-01

    Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(?1,4)GlcUA(?1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-?1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(?1?)UDP [i.e. (GlcNAc?1,4)nGlcNAc(?1?)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of ?4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean ?-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-?1,4)n-GlcNAc(?1?)UDP as HAS reaction products, made in the presence of GlcNAc(?1?)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap. PMID:25583822

  10. Traffic of Chitin Synthase 1 (CHS-1) to the Spitzenkrper and Developing Septa in Hyphae of Neurospora crassa: Actin Dependence and Evidence of Distinct Microvesicle Populations ?

    PubMed Central

    Snchez-Len, Eddy; Verdn, Jorge; Freitag, Michael; Roberson, Robert W.; Bartnicki-Garcia, Salomon; Riquelme, Meritxell

    2011-01-01

    We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkrper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex. PMID:21296914

  11. Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Li, Guo-Tian; Qi, Lin-Lu; Zhang, Yu-Jun; Wang, Chen-Fang; Zhao, Wen-Sheng; Xu, Jin-Rong; Peng, You-Liang

    2012-01-01

    Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases. PMID:22346755

  12. Different chitin synthase genes are required for various developmental and plant infection processes in the rice blast fungus Magnaporthe oryzae.

    PubMed

    Kong, Ling-An; Yang, Jun; Li, Guo-Tian; Qi, Lin-Lu; Zhang, Yu-Jun; Wang, Chen-Fang; Zhao, Wen-Sheng; Xu, Jin-Rong; Peng, You-Liang

    2012-02-01

    Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases. PMID:22346755

  13. An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.

    PubMed

    Ampasala, Dinakar R; Zheng, Sichun; Zhang, Dayu; Ladd, Tim; Doucet, Daniel; Krell, Peter J; Retnakaran, Arthur; Feng, Qili

    2011-02-01

    Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect. PMID:21181720

  14. Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites*

    PubMed Central

    Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

    2012-01-01

    To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xian China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xian D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites. PMID:23024043

  15. Differences among chitin synthase I gene sequences in Trichophyton rubrum and T. violaceum.

    PubMed

    Kano, R; Okabayashi, K; Nakamura, Y; Ooka, S; Kashima, M; Mizoguchi, M; Watanabe, S; Hasegawa, A

    2000-02-01

    Nucleotide sequences of chitin synthase 1 (CHS1) gene were analysed for the phylogenetic relation between Trichophyton violaceum and T. rubrum, including two isolates of T. raubitschekii and one isolate of T. rubrum var. nigricans. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these dermatophytes showed more than 95% similarity between the species. The phylogenetic analysis of their sequences revealed that T. rubrum was genetically distinct from T. violaceum. The specific restriction endonuclease site for HinfI was present in the CHS1 gene sequence of T. rubrum but not in that of T. violaceum. A molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution. PMID:10746227

  16. The chitin synthase genes chsA and chsC are not required for cell wall stress responses in the human pathogen Aspergillus fumigatus

    PubMed Central

    Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen Rao; Lilley, Amanda; Steinbach, William J.

    2013-01-01

    Invasive aspergillosis is a leading cause of mortality in immunocompromised patients. The fungal cell wall is an attractive antifungal target, but it is dynamic and responsive to external stressors. The existence of multiple chitin synthases within Aspergilli is thought to reflect specialized functions in cell wall damage responses that facilitate continued growth and viability. We previously reported increased transcription of Aspergillus fumigatus chitin synthases chsA and chsC following echinocandin treatment, suggesting important roles for these chitin synthases in cell wall compensation. As only partial disruptions have been made of these genes, we generated deletion mutants of chsA and chsC singly (ΔchsA and ΔchsC) and doubly (ΔchsA ΔchsC). The ΔchsA ΔchsC strain displayed reduced total chitin synthase activity. Interestingly, deletion of these chitin synthase genes did not affect levels of chitin or β-1,3-glucan. The ΔchsA, ΔchsC and ΔchsA ΔchsC strains produced wild-type echinocandin-mediated chitin increases, consistent with unaltered cell wall compensation. Furthermore, transcript levels of the remaining chitin synthase genes were unchanged in the mutant strains. Taken together, these results indicate that chsA and chsC do not play a direct role in the cell wall stress response. Our findings support the existence of complex post-transcriptional regulatory mechanisms controlling chitin biosynthetic machinery in response to cell wall damage. PMID:21763289

  17. Molecular identification of Trichophyton rubrum isolate from a dog by chitin synthase 1 (CHS1) gene analysis.

    PubMed

    Kano, R; Hirai, A; Yoshiike, M; Nagata, M; Nakamura, Y; Watanabe, S; Hasegawa, A

    2002-08-01

    A nonsporulating isolate from a dog with dermatophytosis was identified as Trichophyton rubrum by molecular analysis. The nucleotide sequence analysis of the chitin synthase 1 (CHS1) gene from the isolate indicated more than 99% sequence similarity with other human and canine isolates of T rubrum. The molecular typing suggested that isolates of T. rubrum from human and canine sources were genetically identical. PMID:12230227

  18. Bait matrix for delivery of chitin synthesis inhibitors to the formosan subterranean termite (Isoptera: Rhinotermitidae).

    PubMed

    Rojas, M G; Morales-Ramos, J A

    2001-04-01

    The efficacy of three chitin synthesis inhibitors, diflubenzuron, hexaflumuron, and chlorfluazuron, incorporated into a novel bait matrix to kill the Formosan subterranean termite, Coptotermes formosanus Shiraki, was evaluated in the laboratory. The bait matrix was significantly preferred by C. formosanus over southern yellow pine wood in a two-choice feeding test. Bait formulations containing 250 ppm of the three chitin synthesis inhibitors were presented to termite nests with 2,500 individuals (80% workers and 20% soldiers) in the presence of alternative food sources consisting of cardboard and southern yellow pine, Pinus taeda L., wood. None of the bait formulations were significantly repellent or feeding deterrent to the termite workers evidenced by the lack of full consumption of alternative food sources. All nests presented with the bait formulations died within 9 wk, whereas the control nests (bait with no chitin synthesis inhibitors) remained alive 6 mo after the end of the study. No significant differences in consumption were observed among the chitin synthesis inhibitor treatments. Importance of this study for the improvement of current bait technology is discussed. PMID:11332846

  19. Disruption of Spodoptera exigua larval development by silencing chitin synthase gene A with RNA interference.

    PubMed

    Chen, X; Tian, H; Zou, L; Tang, B; Hu, J; Zhang, W

    2008-12-01

    RNA interference (RNAi) is a powerful tool for rapidly analyzing gene functions. However, little is known about the possible use of dsRNA/siRNA as a pest control method. Here, we demonstrate that dsRNA/siRNA can induce the silence of chitin synthase gene A (CHSA), which is an important gene for the growth and development of cuticles and trachea in beet armyworm, Spodoptera exigua. Based on the in vitro RNAi experiments in an insect cell line (Trichoplusia ni High 5), in vivo RNAi was performed by injecting synthesized dsRNA/siRNA into the 4th instar larvae of S. exigua. Significantly lower levels of CHSA transcripts were detected. In addition, the cuticle of these insects was disordered and the epithelial walls of larval trachea did not expand uniformly in injected individuals. Moreover, Injections significantly increased abnormalities relative to control larvae. These results highlighted the possibility of dsRNA/siRNA for gene function studies in lepidopteran insects and future pest control. PMID:18662430

  20. Myosin motor-like domain of class VI chitin synthase CsmB of Aspergillus nidulans is not functionally equivalent to that of class V chitin synthase CsmA.

    PubMed

    Tsuizaki, Makusu; Ohta, Akinori; Horiuchi, Hiroyuki

    2013-01-01

    Chitin is a major cell wall component of many filamentous fungi. Among the eight chitin synthase genes of Aspergillus nidulans, csmA and csmB encode a myosin motor-like domain (MMD) and a chitin synthase domain (CSD) at their N- and C-termini respectively. In our previous reports, we suggested that CsmA and CsmB play compensatory roles essential for polarized hyphal growth although their functions do not completely overlap, and that their MMDs are essential for their functions. In the present study, we constructed chimeric csm genes by swapping N-terminal MMD-encoding halves of csmA and csmB and studied them to identify functional differences in the MMDs. Expression of the chimeric gene encoding the MMD-including half of CsmA (MA) and the CSD-including half of CsmB thoroughly suppressed the phenotypic defects of the ?csmB mutant, whereas the chimeric gene encoding the MMD-including half of CsmB (MB) and the CSD-including half of CsmA did not fully suppress the defects of the ?csmA mutant, suggesting that MA suffices for the function of MB while MB is not functionally equivalent to MA. PMID:23391938

  1. Characterization and functional analysis of a chitin synthase gene (HcCS1) identified from the freshwater pearlmussel Hyriopsis cumingii.

    PubMed

    Zheng, H F; Bai, Z Y; Lin, J Y; Wang, G L; Li, J L

    2015-01-01

    The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain. PMID:26782579

  2. EFFECTS OF 3 CHITIN SYNTHESIS INHIBITORS ON EGG VIABILITY AND SURVIVAL OF COPTOTERMES FORMOSANUS, (ISOPTERA: RHINOTERMITIDAE) INCIPIENT REPRODUCTIVE ADULTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effects of the chitin synthesis inhibitors (CSI), hexaflumuron, diflubenzuron, and lufenuron, on Coptotermes formosanus Shiraki reproductives were studied in the laboratory. Incipient colonies were established by collecting and pairing C. formosanus alates and placing them in dishes containing an ar...

  3. Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

    PubMed Central

    Takeshita, Norio; Wernet, Valentin; Tsuizaki, Makusu; Grn, Nathalie; Hoshi, Hiro-omi; Ohta, Akinori; Fischer, Reinhard; Horiuchi, Hiroyuki

    2015-01-01

    Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinArigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA. PMID:25955346

  4. Molecular cloning, characterization and mRNA expression of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

    PubMed

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-01-01

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

  5. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-01-01

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

  6. Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis.

    PubMed

    Gagou, Mary Elizabeth; Kapsetaki, Manuela; Turberg, Andreas; Kafetzopoulos, Dimitris

    2002-02-01

    Chitin, the major structural polysaccharide of arthropods, is an important constituent of the insect extracellular structures, cuticle and gut peritrophic matrix. Synthesis of cuticular chitin is strictly coordinated with the ecdysone-regulated molting cycle of insect development (the term "ecdysone" is used in this paper instead of "ecdysteroids" since the exact ratio of various hormonal forms changes during metamorphosis). Based on observed similarities between the fungal chitin synthases and other processive beta-glycosyltransferases, we have identified the first insect chitin synthase genes, DmeChSA and DmeChSB (Database accession numbers: EMBL/GenBank/DDBJ A83122, A83126, AJ309488, AJ309489), from Drosophila melanogaster. Chromosomal localization has identified these genes close to and on either side of the centromere of the third chromosome. Partial cDNA clones of both genes have been isolated from a pupal cDNA library. To obtain the first insight into the transcriptional regulation of chitin synthesis, we have monitored the expression of DmeChSA and DmeChSB during the periods of the late-larval and prepupal ecdysone pulses that direct metamorphosis. Transcripts of either gene are barely detected prior to and during the late-larval ecdysone pulse. Once the late-larval ecdysone pulse is ceased completely, both DmeChSA and DmeChSB genes show a remarkable up-regulation. PMID:11755055

  7. Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

    PubMed Central

    Santos, Beatriz; Snyder, Michael

    1997-01-01

    Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p. PMID:9008706

  8. Evolution and Functional Insights of Different Ancestral Orthologous Clades of Chitin Synthase Genes in the Fungal Tree of Life

    PubMed Central

    Li, Mu; Jiang, Cong; Wang, Qinhu; Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2016-01-01

    Chitin synthases (CHSs) are key enzymes in the biosynthesis of chitin, an important structural component of fungal cell walls that can trigger innate immune responses in host plants and animals. Members of CHS gene family perform various functions in fungal cellular processes. Previous studies focused primarily on classifying diverse CHSs into different classes, regardless of their functional diversification, or on characterizing their functions in individual fungal species. A complete and systematic comparative analysis of CHS genes based on their orthologous relationships will be valuable for elucidating the evolution and functions of different CHS genes in fungi. Here, we identified and compared members of the CHS gene family across the fungal tree of life, including 18 divergent fungal lineages. Phylogenetic analysis revealed that the fungal CHS gene family is comprised of at least 10 ancestral orthologous clades, which have undergone multiple independent duplications and losses in different fungal lineages during evolution. Interestingly, one of these CHS clades (class III) was expanded in plant or animal pathogenic fungi belonging to different fungal lineages. Two clades (classes VIb and VIc) identified for the first time in this study occurred mainly in plant pathogenic fungi from Sordariomycetes and Dothideomycetes. Moreover, members of classes III and VIb were specifically up-regulated during plant infection, suggesting important roles in pathogenesis. In addition, CHS-associated networks conserved among plant pathogenic fungi are involved in various biological processes, including sexual reproduction and plant infection. We also identified specificity-determining sites, many of which are located at or adjacent to important structural and functional sites that are potentially responsible for functional divergence of different CHS classes. Overall, our results provide new insights into the evolution and function of members of CHS gene family in the fungal kingdom. Specificity-determining sites identified here may be attractive targets for further structural and experimental studies. PMID:26870058

  9. Evolution and Functional Insights of Different Ancestral Orthologous Clades of Chitin Synthase Genes in the Fungal Tree of Life.

    PubMed

    Li, Mu; Jiang, Cong; Wang, Qinhu; Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2016-01-01

    Chitin synthases (CHSs) are key enzymes in the biosynthesis of chitin, an important structural component of fungal cell walls that can trigger innate immune responses in host plants and animals. Members of CHS gene family perform various functions in fungal cellular processes. Previous studies focused primarily on classifying diverse CHSs into different classes, regardless of their functional diversification, or on characterizing their functions in individual fungal species. A complete and systematic comparative analysis of CHS genes based on their orthologous relationships will be valuable for elucidating the evolution and functions of different CHS genes in fungi. Here, we identified and compared members of the CHS gene family across the fungal tree of life, including 18 divergent fungal lineages. Phylogenetic analysis revealed that the fungal CHS gene family is comprised of at least 10 ancestral orthologous clades, which have undergone multiple independent duplications and losses in different fungal lineages during evolution. Interestingly, one of these CHS clades (class III) was expanded in plant or animal pathogenic fungi belonging to different fungal lineages. Two clades (classes VIb and VIc) identified for the first time in this study occurred mainly in plant pathogenic fungi from Sordariomycetes and Dothideomycetes. Moreover, members of classes III and VIb were specifically up-regulated during plant infection, suggesting important roles in pathogenesis. In addition, CHS-associated networks conserved among plant pathogenic fungi are involved in various biological processes, including sexual reproduction and plant infection. We also identified specificity-determining sites, many of which are located at or adjacent to important structural and functional sites that are potentially responsible for functional divergence of different CHS classes. Overall, our results provide new insights into the evolution and function of members of CHS gene family in the fungal kingdom. Specificity-determining sites identified here may be attractive targets for further structural and experimental studies. PMID:26870058

  10. Dissecting the function of the different chitin synthases in vegetative growth and sexual development in Neurospora crassa.

    PubMed

    Fajardo-Somera, Rosa A; Jhnk, Bastian; Bayram, zgr; Valerius, Oliver; Braus, Gerhard H; Riquelme, Meritxell

    2015-02-01

    Chitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkrper(Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2-GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5-GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages. PMID:25596036

  11. Insight into the adsorption profiles of the Saprolegnia monoica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies.

    PubMed

    Kuang, Guanglin; Liang, Lijun; Brown, Christian; Wang, Qi; Bulone, Vincent; Tu, Yaoquan

    2016-02-10

    The critical role of chitin synthases in oomycete hyphal tip growth has been established. A microtubule interacting and trafficking (MIT) domain was discovered in the chitin synthases of the oomycete model organism, Saprolegnia monoica. MIT domains have been identified in diverse proteins and may play a role in intracellular trafficking. The structure of the Saprolegnia monoica chitin synthase 1 (SmChs1) MIT domain has been recently determined by our group. However, although our in vitro assay identified increased strength in interactions between the MIT domain and phosphatidic acid (PA) relative to other phospholipids including phosphatidylcholine (PC), the mechanism used by the MIT domain remains unknown. In this work, the adsorption behavior of the SmChs1 MIT domain on POPA and POPC membranes was systematically investigated by molecular dynamics simulations. Our results indicate that the MIT domain can adsorb onto the tested membranes in varying orientations. Interestingly, due to the specific interactions between MIT residues and lipid molecules, the binding affinity to the POPA membrane is much higher than that to the POPC membrane. A binding hotspot, which is critical for the adsorption of the MIT domain onto the POPA membrane, was also identified. The lower binding affinity to the POPC membrane can be attributed to the self-saturated membrane surface, which is unfavorable for hydrogen-bond and electrostatic interactions. The present study provides insight into the adsorption profile of SmChs1 and additionally has the potential to improve our understanding of other proteins containing MIT domains. PMID:26818595

  12. Chitin synthase-deficient mutant of Fusarium oxysporum elicits tomato plant defence response and protects against wild-type infection.

    PubMed

    Pareja-Jaime, Yolanda; Martn-Urdroz, Magdalena; Roncero, Mara Isabel Gonzlez; Gonzlez-Reyes, Jos Antonio; Roldn, Mara Del Carmen Ruiz

    2010-07-01

    A mutant of the root pathogen Fusarium oxysporum f. sp. lycopersici, deficient in class V chitin synthase, has been shown previously to be nonvirulent. In this study, we tested the hypothesis that the cause of its avirulence could be the elicitation of the induced plant defence response, leading to the restriction of fungal infection. Co-inoculation of tomato plants with the wild-type strain and the DeltachsV mutant resulted in a significant reduction in symptom development, supporting a protective mechanism exerted by the mutant. The ability of the mutant to penetrate and colonize plant tissues was determined by scanning and transmission electron microscopy, as well as fluorescence microscopy using green fluorescent protein- or cherry fluorescent protein-labelled fungal strains. The extent of wild-type strain colonization in co-inoculated plants decreased steadily throughout the infection process, as shown by the quantification of fungal biomass using real-time polymerase chain reaction. The hypothesis that defence responses are activated by the DeltachsV mutant was confirmed by the analysis of plant pathogenesis-related genes using real-time reverse transcriptase-polymerase chain reaction. Tomato plants inoculated with the DeltachsV mutant showed a three fold increase in endochitinase activity in comparison with wild-type inoculated plants. Taken together, these results suggest that the perturbation of fungal cell wall biosynthesis results in elicitation of the plant defence response during the infection process. PMID:20618706

  13. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter (Oakland, CA); Ring, David B. (Palo Alto, CA); Harrison, Stephen D. (Berkeley, CA); Bray, Andrew M. (Victoria, AU)

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  14. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  15. Farnesyl diphosphate synthase inhibitors with unique ligand-binding geometries.

    PubMed

    Liu, Yi-Liang; Cao, Rong; Wang, Yang; Oldfield, Eric

    2015-03-12

    Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis. PMID:25815158

  16. Antifolate inhibitors of thymidylate synthase as anticancer drugs.

    PubMed

    Jarmu?a, A

    2010-11-01

    Inhibitors of thymidylate synthase (TS) play an essential role in the pharmacological management of several tumors. Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs, with Pemetrexed, unlike Raltitrexed, undergoing further intense clinical development. Other antifolate TS inhibitors, recently/currently tested in clinical studies, that show encouraging anticancer activities are Plevitrexed, GW7904L and Nolatrexed. A new prospect among antifolates, demonstrating a very desirable pattern of pharmacological properties, is BGC 945 that showed promising antitumor activities and has been nominated for clinical development. In this paper, apart from reviewing their biochemical and pharmacological properties, up-to-date characteristics of clinical development of all the mentioned agents are presented. In addition, trends and perspectives for developing improved antifolate inhibitors of TS and future drugs are discussed. Drug resistance is the main barrier to more effective treatment of cancers with antifolates; therefore, mechanisms of antifolate resistance and currently applied approaches to overcome it are also pointed out in the review. PMID:20854257

  17. Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

    PubMed

    Holden, Jeffrey K; Dejam, Dillon; Lewis, Matthew C; Huang, He; Kang, Soosung; Jing, Qing; Xue, Fengtian; Silverman, Richard B; Poulos, Thomas L

    2015-07-01

    Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors. PMID:26062720

  18. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    PubMed Central

    Hargovan, Milan

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors. PMID:24570839

  19. The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

    PubMed

    Gandía, Mónica; Harries, Eleonora; Marcos, Jose F

    2014-06-01

    Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit. PMID:24727399

  20. Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

    PubMed Central

    Walker, Louise A.; Lee, Keunsook K.; Munro, Carol A.

    2015-01-01

    Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections. PMID:26169407

  1. Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion.

    PubMed

    Muszkieta, Laetitia; Aimanianda, Vishukumar; Mellado, Emilia; Gribaldo, Simonetta; Alczar-Fuoli, Laura; Szewczyk, Edyta; Prevost, Marie-Christine; Latg, Jean-Paul

    2014-12-01

    Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA-C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using ?-rec/six system to be able to delete all genes within each family (up to a quadruple ?chsA/C/B/G mutant in family 1 and a quadruple ?csmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs-activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ?chsA, ?chsB and ?chsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs-activity in the ?chsG and ?chsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ?csmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ?csmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (?csmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus. PMID:24946720

  2. Structure-Based Design of Bacterial Nitric Oxide Synthase Inhibitors

    PubMed Central

    2015-01-01

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors. PMID:25522110

  3. Structure-based design of bacterial nitric oxide synthase inhibitors.

    PubMed

    Holden, Jeffrey K; Kang, Soosung; Hollingsworth, Scott A; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B; Poulos, Thomas L

    2015-01-22

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors. PMID:25522110

  4. Structure-based discovery of inhibitors of thymidylate synthase.

    PubMed

    Shoichet, B K; Stroud, R M; Santi, D V; Kuntz, I D; Perry, K M

    1993-03-01

    A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus casei TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK. PMID:8451640

  5. Polar Localizing Class V Myosin Chitin Synthases Are Essential during Early Plant Infection in the Plant Pathogenic Fungus Ustilago maydisW⃞

    PubMed Central

    Weber, Isabella; Aßmann, Daniela; Thines, Eckhard; Steinberg, Gero

    2006-01-01

    Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Δchs6, Δchs7, and Δmcs1 mutants were drastically reduced in virulence. Δmcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Δmcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection. PMID:16314447

  6. Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase

    SciTech Connect

    Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E.

    2010-01-12

    Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

  7. Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris.

    PubMed

    Mommaerts, Veerle; Sterk, Guido; Smagghe, Guy

    2006-08-01

    This research project examined the potential hazards of a major class of insect growth regulators (IGRs) to survival, reproduction and larval growth in bumblebees Bombus terrestris L. Eight chitin synthesis inhibitors (CSIs) were tested: buprofezin, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, lufenuron, novaluron and teflubenzuron. These different IGRs, which are important in the control of pest insects in greenhouses, were applied via three different routes of exposure under laboratory conditions: dermal contact, and orally via the drinking of sugar/water and via pollen. The compounds were tested at their respective maximum field recommended concentrations (MFRC) and also in dose-response assays to calculate LC(50) values. In general, none of the CSIs showed acute worker toxicity. However, there was a dramatic reduction in brood production, especially after oral treatment with pollen and sugar/water. Conspicuously, egg fertility was reduced in all treatments with diflubenzuron and teflubenzuron. In addition to egg mortality, the worker bumblebees removed larvae from the treated nest, and in most cases these individuals were dead first-second instars. Under a binocular microscope, such larvae showed an abnormally formed cuticle leading to mechanical weakness and death. In another series of experiments using (14)C-diflubenzuron and (14)C-flufenoxuron, cuticular penetration in workers was studied for a better understanding of the differences in toxicity. With (14)C-diflubenzuron, transovarial transport and accumulation in the deposited eggs supported the strong reproductive effects. Overall, the present results suggest that CSIs should be applied with caution in combination with bumblebees. The compatibility of each compound to be used in combination with B. terrestris is discussed in relation to calculated LC(50) values, routes of uptake and effects. PMID:16786494

  8. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    PubMed

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

  9. Inhibitors to Polyhydroxyalkanoate (PHA) Synthases: Synthesis, Molecular Docking, and Implications

    PubMed Central

    Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered as an ideal alternative to nonbiodegradable synthetic plastics. However, study of PhaC has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty along with lack of a structure has become the main hurdle to understand and engineer PhaCs for economical PHA production. Here we reported the synthesis of two carbadethia CoA analogs, sT-CH2-CoA 26a and sTet-CH2-CoA 26b as well as sT-aldehyde 29 as new PhaC inhibitors. Study of these analogs with PhaECAv revealed that 26a/b and 29 are competitive and mixed inhibitors, respectively. It was observed that CoA moiety and PHA chain extension can increase binding affinity, which is consistent with the docking study. Estimation from Kic of 26a/b predicts that a CoA analog attached with an octameric-HB chain may facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

  10. Glucosylceramide synthase inhibitors differentially affect expression of glycosphingolipids.

    PubMed

    Alam, Shahidul; Fedier, André; Kohler, Reto S; Jacob, Francis

    2015-04-01

    Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration. PMID:25715344

  11. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  12. Monitoring Chitin Deposition During Septum Assembly in Budding Yeast.

    PubMed

    Arcones, Irene; Roncero, Cesar

    2016-01-01

    The synthesis of the septum is a critical step during cytokinesis in the fungal cell. Moreover, in Saccharomyces cerevisiae septum assembly depends mostly on the proper synthesis and deposition of chitin and, accordingly, on the timely regulation of chitin synthases. In this chapter, we will see how to follow chitin synthesis by two complementary approaches: monitoring chitin deposition in vivo at the septum by calcofluor staining and fluorescence microscopy, and measuring the chitin synthase activities responsible for this synthesis. PMID:26519305

  13. Detection of Microsporum canis in the skin scrapings and hairs of dogs with dermatophytosis based on sequences of the chitin synthase 1 gene.

    PubMed

    Kano, R; Nakamura, Y; Watanabe, S; Hasegawa, A

    2000-01-01

    In the present study, to confirm Microsporum canis infection rapidly, we detected the chitin synthase gene 1 (CHS1) gene of M. canis in the hair and skin samples of four dogs with dermatophytosis. Amplification of the DNAs in the four samples with CHS1 primers yielded fragments of about 620-bp. Nucleotide sequence analysis of the CHS1 gene fragments from samples and a reference strain of M. canis gene showed more than 99% similarity. The method presented in this study can rapidly detect the DNA of M. canis in skin scrapings, and we anticipate that it will be a useful microbiological tool for the diagnosis of M. canis infections in animals and humans. PMID:10981834

  14. Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes Expression and Ultrastructural Hyphal Morphologies

    PubMed Central

    Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

    2013-01-01

    The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely ?-, ?-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage ?-1,3;1,4 glucan synthase celA together with the ?-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

  15. Identification and characterization of inhibitors of Haemophilus influenzae acetohydroxyacid synthase.

    PubMed

    Gedi, Vinayakumar; Moon, Ji-Young; Lim, Won-Mook; Lee, Mi-Young; Lee, Sang-Choon; Koo, Bon-Sung; Govindwar, Sanjay; Yoon, Moon-Young

    2011-06-10

    Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of branched-chain amino acids. The gene coding for the AHAS catalytic subunit from Haemophilus influenzae (Hi) was cloned, overexpressed in Escherichia coli, and purified. To identify new inhibitory scaffolds, we used a high-throughput screen to test 221 small diverse chemical compounds against Hi-AHAS. Compounds were selected for their ability to inhibit AHAS in vitro. The screen identified 3 compounds, each representing a structural class, as Hi-AHAS inhibitors with an IC(50) in the low micromolar range (4.4-14.6 μM). The chemical scaffolds of the three compounds were oxa-1-thia-4-aza-cyclopenta[b]naphthalene (KHG25229), phenyl-2,3-dihydro-isothiazole (KHG25386), and phenyl-pyrrolidine-3-carboxylic acid phenylamide (KHG25056). Further, molecular docking of the two most potent chemicals, KHG25229 and KHG25386, in Hi-AHAS yielded binding energies of -10.41 and -9.21 kcal/mol, respectively. The binding modes were consistent with inhibition mechanisms, as both chemicals oriented outside the active site. As the need for novel antibiotic classes to combat drug resistant bacteria increases, screening compounds that act against Hi-AHAS may assist in the identification of potential new anti-Hi drugs. PMID:22112263

  16. Chitin in Diatoms and Its Association with the Cell Wall ?

    PubMed Central

    Durkin, Colleen A.; Mock, Thomas; Armbrust, E. Virginia

    2009-01-01

    Chitin is a globally abundant polymer widely distributed throughout eukaryotes that has been well characterized in only a few lineages. Diatoms are members of the eukaryotic lineage of stramenopiles. Of the hundreds of diatom genera, two produce long fibers of chitin that extrude through their cell walls of silica. We identify and describe here genes encoding putative chitin synthases in a variety of additional diatom genera, indicating that the ability to produce chitin is more widespread and likely plays a more central role in diatom biology than previously considered. Diatom chitin synthases fall into four phylogenetic clades. Protein domain predictions and differential gene expression patterns provide evidence that chitin synthases have multiple functions within a diatom cell. Thalassiosira pseudonana possesses six genes encoding three types of chitin synthases. Transcript abundance of the gene encoding one of these chitin synthase types increases when cells resume division after short-term silicic acid starvation and during short-term limitation by silicic acid or iron, two nutrient conditions connected in the environment and known to affect the cell wall. During long-term silicic acid starvation transcript abundance of this gene and one additional chitin synthase gene increased at the same time a chitin-binding lectin localized to the girdle band region of the cell wall. Together, these results suggest that the ability to produce chitin is more widespread in diatoms than previously thought and that a subset of the chitin produced by diatoms is associated with the cell wall. PMID:19429777

  17. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  18. Spitzenkrper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa?

    PubMed Central

    Riquelme, Meritxell; Bartnicki-Garca, Salomon; Gonzlez-Prieto, Juan Manuel; Snchez-Len, Eddy; Verdn-Ramos, Jorge A.; Beltrn-Aguilar, Alejandro; Freitag, Michael

    2007-01-01

    The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkrper (Spk). In distal regions (beyond 40 ?m from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 ?m from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 ?m from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway. PMID:17644657

  19. Behavioral and histological changes in the Formosan subterranean termite (Isoptera: Rhinotermitidae) induced by the chitin synthesis inhibitor noviflumuron.

    PubMed

    Xing, Lin; Chouvenc, Thomas; Su, Nan-Yao

    2014-04-01

    This study describes the behavioral and histological changes of the molting process in Coptotermes formosanus Shiraki caused by the chitin synthesis inhibitor noviflumuron. Termites exposed to noviflumuron initiated ecdysis as untreated individuals did; however, peristalsis contractions were weak and the expansion of the dorsal breach of the exoskeleton did not occur. Treated termites could not complete their molting process and died after the initiation of the ecdysis. Histological observations showed that the process of voiding the gut protozoa during premolting was not affected by the noviflumuron treatment. However, the formation of the new cuticle was disrupted resulting in the loss of integrity of the cuticle. The alteration of the cuticle was visible in the gizzard (foregut), the thoracic pleurons, and most of the exoskeleton. Muscles were partially able to reattach to the incompletely formed new cuticle, and muscle contractions resulted in tearing off the cuticle. Because the integrity of the newly formed cuticle was compromised by the noviflumuron treatment, we concluded that termites' death was caused primarily by the loss of hemolymph as a result of the damage done by the muscle contractions on the exoskeleton during the peristalsis. As the physiological homeostasis was disrupted, termites were too weak to shed their old cuticle, ultimately resulting in termite dying during the molting process. PMID:24772556

  20. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    PubMed Central

    Lee, Chia-Hsien; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2011-01-01

    Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR) method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs. PMID:21954360

  1. Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase

    SciTech Connect

    Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

    1986-05-01

    Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

  2. A high-throughput screen utilizing the fluorescence of riboflavin for identification of lumazine synthase inhibitors.

    PubMed

    Chen, Jinhua; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Haase, Ilka; Georg, Gunda; Ye, Qi-Zhuang; Ma, Zeqiang; Cushman, Mark

    2005-03-01

    A high-throughput screening method based on the competitive binding of a lumazine synthase inhibitor and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase was developed. This assay is sensitive, simple, and robust. During assay development, all of the known active inhibitors tested were positively identified. Preliminary high-throughput screening in 384-well format resulted in a Z factor of 0.7. The approach utilizes a thermodynamic assay to bypass the problems associated with the instabilities of both lumazine synthase substrates that complicate the use of a kinetic assay in a high-throughput format, and it removes the time element from the assay, thus simplifying the procedure. PMID:15707942

  3. ECOLOGICAL FITNESS OF ACETOLACTATE SYNTHASE INHIBITORRESISTANT AND SUSCEPTIBLE DOWNY BROME (BROMUS TECTORUM) BIOTYPES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies were conducted to determine the relative fitness and competitive ability of an acetolactate synthase (ALS) inhibitorresistant (R) downy brome biotype compared with a susceptible (S) biotype. In previous research, the mechanism of resistance was determined to be an altered ALS enzyme. Seed g...

  4. MULTI-ANALYTE CHEMISTRY METHODS FOR PESTICIDES WHICH ARE ACETOLACTATE SYNTHASE (ALS) INHIBITORS IN SOIL

    EPA Science Inventory

    A joint EPA/state/industry working group has developed several multi-analyte methods to analyze soils for low ppb (parts per billion) levels of herbicides (such as sulfonylureas, imidazolinones, and sulfonamides) that are acetolactate synthase (ALS) inhibitors and may cause phyto...

  5. The Impact of Selection with Diflubenzuron, a Chitin Synthesis Inhibitor, on the Fitness of Two Brazilian Aedes aegypti Field Populations.

    PubMed

    Belinato, Thiago Affonso; Valle, Denise

    2015-01-01

    Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators. PMID:26107715

  6. Effect of triflumuron, a chitin synthesis inhibitor, on Aedes aegypti, Aedes albopictus and Culex quinquefasciatus under laboratory conditions

    PubMed Central

    2013-01-01

    Background Resistance to traditional insecticides represents a threat to the control of disease vectors. The insect growth regulators (IGR) are a potential alternative to control mosquitoes, including resistant populations. The chitin synthesis inhibitors (CSI) are IGRs, which interfere with the insect molting process and represent one major class of compounds against Aedes aegypti populations resistant to the larvicide organophosphate temephos. In the present study, we evaluated the efficacy of the CSI triflumuron on Culex quinquefasciatus, Aedes albopictus and against several Ae. aegypti field populations. Methods The efficacy of triflumuron, against Cx. quinquefasciatus and Ae. albopictus was evaluated with laboratory strains through doseresponse assays. Additionaly, this CSI was tested against seven Ae. aegypti field populations exhibiting distinct resistance levels to both temephos and the pyrethroid deltamethrin. Aedes aegypti populations were exposed to both a dose that inhibits 99% of the adult emergence of mosquitoes from the susceptible reference strain, Rockefeller, (EI99?=?3.95??g/L) and the diagnostic dose (DD), corresponding to twice the EI99. Results Our results indicate that triflumuron was effective in emergence inhibition (EI) of Cx. quinquefasciatus (EI50= 5.28??g/L; EI90= 12.47??g/L) and Ae. albopictus (EI50= 1.59??g/L; EI90= 2.63??g/L). Triflumuron was also effective against seven Ae. aegypti Brazilian populations resistant to both temephos and deltamethrin. Exposure of all the Ae. aegypti populations to the triflumuron EI99 of the susceptible reference strain, Rockefeller, resulted in complete inhibition of adult emergence, suggesting no cross-resistance among traditional insecticides and this CSI. However, a positive correlation between temephos resistance and tolerance to triflumuron was observed. Conclusion The results suggest that triflumuron represents a potential tool for the control of disease vectors in public health. Nevertheless, they point to the need of constant monitoring of the susceptibility status of vector populations to CSIs. PMID:23557173

  7. The Impact of Selection with Diflubenzuron, a Chitin Synthesis Inhibitor, on the Fitness of Two Brazilian Aedes aegypti Field Populations

    PubMed Central

    Belinato, Thiago Affonso; Valle, Denise

    2015-01-01

    Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators. PMID:26107715

  8. Temporal Phosphoproteome Dynamics Induced by an ATP Synthase Inhibitor Citreoviridin.

    PubMed

    Hu, Chia-Wei; Hsu, Chia-Lang; Wang, Yu-Chao; Ishihama, Yasushi; Ku, Wei-Chi; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-12-01

    Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 ? and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies. PMID:26503892

  9. Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera dorsalis

    PubMed Central

    Yang, Wen-Jia; Xu, Kang-Kang; Cong, Lin; Wang, Jin-Jun

    2013-01-01

    Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5?- and 3?-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E. PMID:23569438

  10. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis.

    PubMed

    Yang, Wen-Jia; Xu, Kang-Kang; Cong, Lin; Wang, Jin-Jun

    2013-01-01

    Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5'- and 3'-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E. PMID:23569438

  11. Structural characterization of substrate and inhibitor binding to farnesyl pyrophosphate synthase from Pseudomonas aeruginosa

    PubMed Central

    Schmidberger, Jason W.; Schnell, Robert; Schneider, Gunter

    2015-01-01

    Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0 Å. The enzyme shows the typical α-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme–ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes. PMID:25760619

  12. Fumonisins and fumonisin analogs as inhibitors of ceramide synthase and inducers of apoptosis.

    PubMed

    Desai, Kena; Sullards, M Cameron; Allegood, Jeremy; Wang, Elaine; Schmelz, Eva M; Hartl, Michaela; Humpf, Hans-Ulrich; Liotta, D C; Peng, Qiong; Merrill, Alfred H

    2002-12-30

    Sphingoid bases are growth inhibitory and pro-apoptotic for many types of cells when added to cells exogenously, and can be elevated to toxic amounts endogenously when cells are exposed to inhibitors of ceramide synthase. An important category of naturally occurring inhibitors are the fumonisins, which inhibit ceramide synthase through structural similarities with both the sphingoid base and fatty acyl-CoA co-substrates. Fumonisins cause a wide spectrum of disease (liver and renal toxicity and carcinogenesis, neurotoxicity, induction of pulmonary edema, and others), and most-possibly all-of the pathophysiologic effects of fumonisins are attributable to disruption of the sphingolipid metabolism. The products of alkaline hydrolysis of fumonisins (which occurs during the preparation of masa flour for tortillas) are aminopentols that also inhibit ceramide synthase, but more weakly. Nonetheless, the aminopentols (and other 1-deoxy analogs of sphinganine) are acylated to derivatives that inhibit ceramide synthase, perhaps as product analogs, elevate sphinganine, and kill the cells. Somewhat paradoxically, fumonisins sometimes stimulate growth and inhibit apoptosis, possibly due to elevation of sphinganine 1-phosphate, which is known to have these cellular effects. These findings underscore the complexity of sphingolipid metabolism and the difficulty of identifying the pertinent mediators unless a full profile of the potentially bioactive species is evaluated. PMID:12531553

  13. U-19451A: a selective inducible nitric oxide synthase inhibitor.

    PubMed

    Stratman, N C; Fici, G J; Sethy, V H

    1996-01-01

    Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock. Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures. Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus gamma-interferon were used for iNOS activity. Rat cerebellum and primary cerebellar granule cell cultures were utilized for neuronal NOS (nNOS) activity. S-methylthiourea (SMT) and L-nitroarginine methyl ester (L-NAME), selective iNOS and nNOS inhibitors, respectively, were chosen as standards. Both U-19451A and SMT were 4-times more selective for iNOS as compared to nNOS in tissues. U-19451A was more selective than SMT for iNOS inhibition using cultures. L-NAME was 16-31 times more selective for inhibiting nNOS activity. Based on the selectivity of U-19451A for iNOS inhibition, this drug would be expected to be effective in the treatment of diseases with inflammatory pathology without producing side effects associated with nNOS inhibition. PMID:8795706

  14. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    PubMed

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies. PMID:11827032

  15. Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

    PubMed Central

    Richie, Daryl L.; Thompson, Katherine V.; Studer, Christian; Prindle, Vivian C.; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A.; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N. Rao; Tallarico, John A.

    2013-01-01

    High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. PMID:23478965

  16. Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

    PubMed Central

    Bergstrom, J D; Kurtz, M M; Rew, D J; Amend, A M; Karkas, J D; Bostedor, R G; Bansal, V S; Dufresne, C; VanMiddlesworth, F L; Hensens, O D

    1993-01-01

    Three closely related fungal metabolites, zaragozic acids A, B, and C, that are potent inhibitors of squalene synthase have been isolated and characterized. Zaragozic acids A, B, and C were produced from an unidentified sterile fungal culture, Sporormiella intermedia, and Leptodontium elatius, respectively. The structures of the zaragozic acids and their trimethyl esters were determined by a combination of physical and chemical techniques. The zaragozic acids are characterized by a novel 2,8-dioxobicyclo[3.2.1]octane-4,6,7- trihydroxyl-3,4,5-tricarboxylic acid core and differ from each other in the structures of the 6-acyl and 1-alkyl side chains. They were found to be potent competitive inhibitors of rat liver squalene synthase with apparent Ki values of 78 pM, 29 pM, and 45 pM, respectively. They inhibited cholesterol synthesis in Hep G2 cells, and zaragozic acid A was an inhibitor of acute hepatic cholesterol synthesis in the mouse (50% inhibitory dose of 200 micrograms/kg of body weight). Inhibition of squalene synthase in cells and in vivo was accompanied by an accumulation of label from [3H]mevalonate into farnesyl diphosphate, farnesol, and organic acids. These data indicate that the zaragozic acids are a previously unreported class of therapeutic agents with potential for the treatment of hypercholesterolemia. PMID:8419946

  17. Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

    PubMed

    Richie, Daryl L; Thompson, Katherine V; Studer, Christian; Prindle, Vivian C; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N Rao; Tallarico, John A; Ryder, Neil S; Hoepfner, Dominic

    2013-05-01

    High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. PMID:23478965

  18. Prodrugs of thymidylate synthase inhibitors: potential for antibody directed enzyme prodrug therapy (ADEPT).

    PubMed

    Springer, C J; Bavetsias, V; Jackman, A L; Boyle, F T; Marshall, D; Pedley, R B; Bisset, G M

    1996-12-01

    Prodrugs of quinazoline antifolate thymidylate synthase (TS) inhibitors have been designed and synthesized for use in antibody-directed enzyme prodrug therapy (ADEPT). The syntheses of the alpha-linked dipeptides of two potent thymidylate synthase inhibitors, ZD1694 [N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid] and ICI198583 ¿N-[4-[N-[(2-methyl-3,4-dihydro-4-oxo-6-quinazolinyl) methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid¿ are described. The alpha-carboxyl of the glutamic acid has been linked through an amide bond to an L-alanine or an L-glutamic acid. The alpha-linked L-dipeptide prodrugs were designed to be activated to their corresponding thymidylate synthase inhibitors at a tumour site by prior administration of a monoclonal antibody conjugated to the enzyme carboxypeptidase A (CPA). The viability of a colorectal cell line was monitored with the potential prodrugs in the presence or absence of CPA or with the parent drugs alone. All the dipeptides had greatly decreased cytotoxicity, with a deactivation of approximately 100-fold for the ZD1694 prodrugs and approximately 20-200-fold for the ICI198583 prodrugs. Activation of the alpha-linked L-alanine dipeptides with CPA led to a cytotoxicity enhancement of approximately 10-100 fold. PMID:9022750

  19. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    SciTech Connect

    Morgunova, Ekaterina; Illarionov, Boris; Saller, Sabine; Popov, Aleksander; Sambaiah, Thota; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 . The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  20. Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases.

    PubMed

    Adada, Mohamad; Luberto, Chiara; Canals, Daniel

    2016-05-01

    Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann-Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics. PMID:26200918

  1. Exploration of Peptide Inhibitors of Human Squalene Synthase through Molecular Modeling and Phage Display Technique.

    PubMed

    Shiuan, David; Lin, Hwan-Kang; Chen, Yue-Hao; Chang, Ding-Kwo; Huang, Kao-Jean; Farh, Lynn

    2016-01-01

    Many studies have demonstrated the role of elevated levels of serum cholesterol in the pathogenesis of atherosclerosis and coronary heart disease. Various drugs targeting the key enzymes involved in the cholesterol biosynthesis pathway have been investigated for the treatment of hypercholesterolemia. Human squalene synthase has been one of the most important targets for therapeutic intervention. In the present study, we used the recombinant human squalene synthase as the lure for screening the peptide inhibitors from phage-displayed random peptide library. The tightly bound phages and their derived peptides were further evaluated based on their potential binding capabilities, molecular modeling characteristics and predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. Several hexa-peptides and tetra-peptides were finally synthesized to assay their inhibitory effects toward the recombinant human squalene synthase. The results demonstrated that the hexa-peptide FTACNW and tetra-peptide VACL can inhibit human squalene synthase effectively (with IC50 values near 100 μM) and may have potential to develop further as future hypocholesterolemia agents. PMID:26438313

  2. Phosphonosulfonates Are Potent, Selective Inhibitors of Dehydrosqualene Synthase and Staphyloxanthin Biosynthesis in Staphylococcus aureus

    PubMed Central

    Song, Yongcheng; Lin, Fu-Yang; Yin, Fenglin; Hensler, Mary; Poveda, Carlos A. Rodrígues; Mukkamala, Dushyant; Cao, Rong; Wang, Hong; Morita, Craig T.; Pacanowska, Dolores González; Nizet, Victor; Oldfield, Eric

    2009-01-01

    Staphylococcus aureus produces a golden carotenoid virulence factor called staphyloxanthin (STX), and we report here the inhibition of the enzyme, dehydrosqualene synthase (CrtM), responsible for the first committed step in STX biosynthesis. The most active compounds are halogen-substituted phosphonosulfonates, with Ki values as low as 5 nM against the enzyme and IC50 values for STX inhibition in S. aureus as low as 11 nM. There is, however, only a poor correlation (R2 = 0.27) between enzyme and cell pIC50 (= −log10 IC50) values. The ability to predict cell from enzyme data improves considerably (to R2 = 0.72) with addition of two more descriptors. We also investigated the activity of these compounds against human squalene synthase (SQS), as a counterscreen, finding several potent STX biosynthesis inhibitors with essentially no squalene synthase activity. These results open up the way to developing potent and selective inhibitors of an important virulence factor in S. aureus, a major human pathogen. PMID:19191557

  3. Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors.

    PubMed

    van den Berg, Richard J B H N; van Rijssel, Erwin R; Ferraz, Maria Joao; Houben, Judith; Strijland, Anneke; Donker-Koopman, Wilma E; Wennekes, Tom; Bonger, Kimberly M; Ghisaidoobe, Amar B T; Hoogendoorn, Sascha; van der Marel, Gijsbert A; Code, Jeroen D C; Overkleeft, Herman S; Aerts, Johannes M F G

    2015-12-01

    Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type?2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200?nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator. PMID:26492941

  4. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia.

    PubMed

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2015-11-25

    The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  5. Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa.

    PubMed

    Orenes Lorente, Silvia; Gómez, Rosario; Jiménez, Carmen; Cammerer, Simon; Yardley, Vanessa; de Luca-Fradley, Kate; Croft, Simon L; Ruiz Perez, Luis M; Urbina, Julio; Gonzalez Pacanowska, Dolores; Gilbert, Ian H

    2005-05-16

    In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved. PMID:15848765

  6. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    PubMed Central

    Morgunova, Ekaterina; Illarionov, Boris; Saller, Sabine; Popov, Aleksander; Sambaiah, Thota; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2010-01-01

    The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R cryst = 23.7% (R free = 28.4%) at a resolution of 3.5?. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis. PMID:20823551

  7. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis.

    PubMed

    Morgunova, Ekaterina; Illarionov, Boris; Saller, Sabine; Popov, Aleksander; Sambaiah, Thota; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2010-09-01

    The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R(cryst) = 23.7% (R(free) = 28.4%) at a resolution of 3.5 A. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis. PMID:20823551

  8. ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

    PubMed Central

    Hong, Sangjin; Pedersen, Peter L.

    2008-01-01

    Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  9. Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

    PubMed

    Skovpen, Yulia V; Conly, Cuylar J T; Sanders, David A R; Palmer, David R J

    2016-02-17

    Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development. PMID:26836694

  10. Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.

    PubMed

    Ghisaidoobe, Amar T; van den Berg, Richard J B H N; Butt, Saleem S; Strijland, Anneke; Donker-Koopman, Wilma E; Scheij, Saskia; van den Nieuwendijk, Adrianus M C H; Koomen, Gerrit-Jan; van Loevezijn, Arnold; Leemhuis, Mark; Wennekes, Tom; van der Stelt, Mario; van der Marel, Gijsbert A; van Boeckel, Constant A A; Aerts, Johannes M F G; Overkleeft, Herman S

    2014-11-13

    This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics. PMID:25250725

  11. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    PubMed Central

    McMillan, Kirk; Adler, Marc; Auld, Douglas S.; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.; Ohlmeyer, Michael H. J.; Pan, Gonghua; Parkinson, John F.; Phillips, Gary B.; Polokoff, Mark A.; Sigal, Nolan H.; Vergona, Ronald; Whitlow, Marc; Young, Tish A.; Devlin, James J.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies. PMID:10677491

  12. Symmetric Double-Headed Aminopyridines, A Novel Strategy for Potent and Membrane-Permeable Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Xue, Fengtian; Fang, Jianguo; Delker, Silvia L.; Li, Huiying; Martsek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2011-01-01

    We report novel neuronal nitric oxide synthase (nNOS) inhibitors based on a symmetric double-headed aminopyridine scaffold. The inhibitors were designed from crystal structures of leads 1 and 2 (Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman, R. B.; Poulos, T. L. Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of cerebral palsy. J. Am. Chem. Soc. 2010, 132, 54375442) and synthesized using a highly efficient route. The best inhibitor, 3j, showed low nanomolar inhibitory potency and modest isoform selectivity. It also exhibited enhanced membrane permeability. Inhibitor 3j binds to both the substrate site and the pterin site in nNOS but only to the substrate site in eNOS. These compounds provide a basis for further development of novel, potent, isoform selective, and bioavailable inhibitors for nNOS. PMID:21410186

  13. Synthesis and biological evaluation of nonsymmetrical aromatic disulfides as novel inhibitors of acetohydroxyacid synthase.

    PubMed

    Li, Zai-Shun; Wang, Wei-Min; Lu, Wei; Niu, Cong-Wei; Li, Yong-Hong; Li, Zheng-Ming; Wang, Jian-Guo

    2013-07-01

    46 Novel nonsymmetrical aromatic disulfides containing [1,3,4]thiadiazole or [1,3,4]oxadiazole groups were synthesized and their biological activities were evaluated as inhibitors of acetohydroxyacid synthase (AHAS, EC 2.2.1.6). Besides their strong in vitro inhibition against plant AHAS, compounds 3e and 3f also display 80-100% post-emergence herbicidal activities in greenhouse bioassay at 1500g /ha dosage. The assay of exogenous branched-chain amino acids supplementation on rape root growth of 3e suggests that the herbicidal activity has relationship with AHAS inhibition. PMID:23726033

  14. Anmindenols A and B, inducible nitric oxide synthase inhibitors from a marine-derived Streptomyces sp.

    PubMed

    Lee, Jihye; Kim, Hiyoung; Lee, Tae Gu; Yang, Inho; Won, Dong Hwan; Choi, Hyukjae; Nam, Sang-Jip; Kang, Heonjoong

    2014-06-27

    Anmindenols A (1) and B (2), inhibitors of inducible nitric oxide synthase (iNOS), were isolated from a marine-derived bacterium Streptomyces sp. Their chemical structures were elucidated by interpreting various spectroscopic data, including IR, MS, and NMR. Anmindenols A and B are sesquiterpenoids possessing an indene moiety with five- and six-membered rings derived from isoprenyl units. The absolute configuration of C-4 in anmindenol B was determined by electronic circular dichroism (ECD) of a dimolybdenum complex. Anmindenols A (1) and B (2) inhibited nitric oxide production in stimulated RAW 264.7 macrophage cells with IC50 values of 23 and 19 μM, respectively. PMID:24878306

  15. Potent, Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Xue, Fengtian; Li, Huiying; Delker, Silvia L.; Fang, Jianguo; Martsek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) vs. (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors we have introduced a CF2 moiety geminal to an amino group in the long tail of one of these inhibitors, which reduced its basicity, resulting in compounds with monocationic character under physiological pH conditions. Biological evaluations have led to a nNOS inhibitor with a Ki of 36 nM and high selectivity for nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pKa NH is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially no oral bioavailability for the parent compound. This indicates that the goal of this research to make compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability, but which can become protonated when bound to NOS, has been accomplished. PMID:20843082

  16. Chitin Research Revisited

    PubMed Central

    Khoushab, Feisal; Yamabhai, Montarop

    2010-01-01

    Two centuries after the discovery of chitin, it is widely accepted that this biopolymer is an important biomaterial in many aspects. Numerous studies on chitin have focused on its biomedical applications. In this review, various aspects of chitin research including sources, structure, biosynthesis, chitinolytic enzyme, chitin binding protein, genetic engineering approach to produce chitin, chitin and evolution, and a wide range of applications in bio- and nanotechnology will be dealt with. PMID:20714419

  17. Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

    PubMed Central

    Begley, Darren W.; Edwards, Thomas E.; Raymond, Amy C.; Smith, Eric R.; Hartley, Robert C.; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D.; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

    2011-01-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme fornucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, isreported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

  18. Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.

    PubMed

    Begley, Darren W; Edwards, Thomas E; Raymond, Amy C; Smith, Eric R; Hartley, Robert C; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D; Myler, Peter J; Staker, Bart L; Stewart, Lance J

    2011-09-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

  19. Fatty acid synthase inhibitor cerulenin suppresses liver metastasis of colon cancer in mice.

    PubMed

    Murata, Soichiro; Yanagisawa, Kazuhiko; Fukunaga, Kiyoshi; Oda, Tatsuya; Kobayashi, Akihiko; Sasaki, Ryoko; Ohkohchi, Nobuhiro

    2010-08-01

    Fatty acid synthase (FAS) is highly expressed in many kinds of human cancers, including colorectal cancer (CRC), and we have investigated the potential use of FAS inhibitors for chemoprevention of liver metastasis of CRC in mice. Expression of FAS was evaluated in murine CRC cell lines Colon 26 and CMT 93. Cerulenin, a natural inhibitor of FAS, induced apoptosis in these cell lines. The ability of cerulenin to prevent development of liver metastatic lesions in Colon 26 was evaluated. The numbers and sizes of liver metastatic CRC tumors were significantly reduced by treating mice with cerulenin. Cerulenin treatment was associated with reduced levels of phosphorylated Akt in Colon 26 cells, suggesting that inhibition of this signal transduction pathway might be involved in the chemopreventive activity of this compound. Based on studies in mouse models, inhibiting FAS would be an effective strategy to prevent and retard growth of liver metastatic tumors of CRC that have high expression of this enzyme. PMID:20491775

  20. High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors

    PubMed Central

    Takahashi, Sachiko; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Sato, Masaru; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Tanaka, Hiroaki; Urade, Yoshihiro

    2010-01-01

    Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D2, an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC50) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC50 value of 50?nM extended to 1.1? resolution at 100?K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein. PMID:20606289

  1. Development Of Nitric Oxide Synthase Inhibitors for Neurodegeneration and Neuropathic Pain

    PubMed Central

    Mukherjee, Paramita; Cinelli, Maris A.; Kang, Soosung; Silverman, Richard B.

    2014-01-01

    Nitric oxide (NO) is an important signaling molecule in the human body, playing a crucial role in cell and neuronal communication, regulation of blood pressure, and in immune activation. However, overproduction of NO by the neuronal isoform of nitric oxide synthase (nNOS)is one of the fundamental causes underlying neurodegenerative disorders and neuropathic pain. Therefore, developing small molecules for selective inhibition of nNOS over related isoforms(eNOS and iNOS) is therapeutically desirable. The aims of this review focus on the regulation and dysregulation of NO signaling, the role of NO in neurodegeneration and pain, the structure and mechanism of nNOS, and the use of this information to design selective inhibitors of this enzyme. Structure-based drug design, the bioavailability and pharmacokinetics of these inhibitors, and extensive target validation through animal studies are addressed. PMID:24549364

  2. Localized deposition of chitin on the yeast cell surface in response to mating pheromone

    PubMed Central

    Schekman, Randy; Brawley, Vicki

    1979-01-01

    Treatment of a mating-type Saccharomyces cerevisiae cells with the pheromone ?-factor (secreted by ? mating-type cells) induces the synthesis of chitin. Small daughter cells, which start with no detectable chitin, make 3 times more chitin when grown in the presence of ?-factor than do untreated exponentially growing cells. Budding cells accumulate chitin in the nascent division septum [Cabib, E. & Bowers, B. (1975) J. Bacteriol. 124, 1586), as detected by staining with the fluorescent dye primulin. In the absence of a division septum, ?-factor-treated cells accumulate chitin in the area of pheromone-stimulated growth. Enzymatic lysis of budding and pheromone-treated cells allows the separation of membrane-bound chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-?-acetamidodeoxyglucosyltransferase, EC 2.4.1.16) activity from a dense particulate fraction containing chitin. Chitin synthase activity is associated with both the plasma membrane and small intracellular particles. During pheromone treatment, the levels of chitin synthase in the plasma membrane and in intracellular particle fractions increase 11- and 4-fold, respectively. Although chitin synthase is made as zymogen that requires proteolytic activation, the plasma membrane of pheromone-treated cells shows a significant fraction of preactivated enzyme; intracellular membrane-bound synthase is found exclusively in the zymogen form. Images PMID:16592617

  3. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases

    PubMed Central

    Christensen, Quin H.; Grove, Tyler L.; Booker, Squire J.; Greenberg, E. Peter

    2013-01-01

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 M and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, antiquorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach. PMID:23924613

  4. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.

    PubMed

    Christensen, Quin H; Grove, Tyler L; Booker, Squire J; Greenberg, E Peter

    2013-08-20

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti-quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach. PMID:23924613

  5. Identification and Characterization of Novel Microsomal Prostaglandin E Synthase-1 Inhibitors for Analgesia.

    PubMed

    Chandrasekhar, Srinivasan; Harvey, Anita K; Yu, Xiao-Peng; Chambers, Mark G; Oskins, Jennifer L; Lin, Chaohua; Seng, Thomas W; Thibodeaux, Stefan J; Norman, Bryan H; Hughes, Norman E; Schiffler, Matthew A; Fisher, Matthew J

    2016-03-01

    Prostaglandin (PG) E2 plays a critical role in eliciting inflammation. Nonsteroidal anti-inflammatory drugs and selective inhibitors of cyclooxygenase, which block PGE2 production, have been used as key agents in treating inflammation and pain associated with arthritis and other conditions. However, these agents have significant side effects such as gastrointestinal bleeding and myocardial infarction, since they also block the production of prostanoids that are critical for other normal physiologic functions. Microsomal prostaglandin E2 synthase-1 is a membrane-bound terminal enzyme in the prostanoid pathway, which acts downstream of cyclooxygenase 2 and is responsible for PGE2 production during inflammation. Thus, inhibition of this enzyme would be expected to block PGE2 production without inhibiting other prostanoids and would provide analgesic efficacy without the side effects. In this report, we describe novel microsomal prostaglandin E2 synthase-1 inhibitors that are potent in blocking PGE2 production and are efficacious in a guinea pig monoiodoacetate model of arthralgia. These molecules may be useful in treating the signs and symptoms associated with arthritis. PMID:26740668

  6. Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

    PubMed

    Marzinzik, Andreas L; Amstutz, Ren; Bold, Guido; Bourgier, Emmanuelle; Cotesta, Simona; Glickman, J Fraser; Gtte, Marjo; Henry, Christelle; Lehmann, Sylvie; Hartwieg, J Constanze D; Ofner, Silvio; Pell, Xavier; Roddy, Thomas P; Rondeau, Jean-Michel; Stauffer, Frdric; Stout, Steven J; Widmer, Armin; Zimmermann, Johann; Zoller, Thomas; Jahnke, Wolfgang

    2015-11-01

    Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases. PMID:26381451

  7. Recent Advances in the Development of Undecaprenyl Pyrophosphate Synthase Inhibitors as Potential Antibacterials.

    PubMed

    Jukic, Marko; Rozman, Kaja; Gobec, Stanislav

    2016-01-01

    Expanding antibiotic use in clinical practice and emergence of bacterial resistance are fueling research efforts for the development of novel antibacterials. Underexploited or completely novel mechanistic approaches and biological targets are of especial interest. Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in the biosynthesis of the bacterial cell wall. Although UppS is a validated target, no selective inhibitors occur in materia medica. Nevertheless, several native substrate analogues have been reported and used in enzyme kinetics studies or as pharmacological probes. The majority of small-molecule UppS inhibitors belong to the well-known class of bisphosphonates that are primarily used for treatment of bone resorption disorders. The most potent compound of this class has an IC50 of 0.59 M. Inherently, the selectivity and suitability of such compounds for antimicrobial drug design can be questioned. Therefore, highthroughput and virtual screenings for non-bisphosphonate inhibitors were performed, and nanomolar inhibitors of UppS were identified, some with antimicrobial activities towards clinically relevant strains. The reported scaffolds belong to tetramic and tetronic acids with IC50 in the 100-nM range, and to dihydropyridines with IC50 down to 40 nM, all with antibacterial activity. Aryl-diketo acids are also potent inhibitors with MRSA antimicrobial activity, with the allosteric inhibitor methylisoxazole-4-carboxamide (IC50, 50 nM) active on several pathogenic Streptococcus pneumoniae strains. Clomiphene is a well-known oestrogen receptor modulator, and it has been reported to inhibit UppS. Although conclusions on the structure activity relationships cannot be drawn from all these data, these compound series represent an important contribution to the field of antibiotics. PMID:26718796

  8. Crystal structure of an archaeal pentameric riboflavin synthase in complex with a substrate analog inhibitor: stereochemical implications.

    PubMed

    Ramsperger, Arne; Augustin, Martin; Schott, Ann-Kathrin; Gerhardt, Stefan; Krojer, Tobias; Eisenreich, Wolfgang; Illarionov, Boris; Cushman, Mark; Bacher, Adelbert; Huber, Robert; Fischer, Markus

    2006-01-13

    Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers. PMID:16272154

  9. Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

    PubMed

    Haeuptle, Micha A; Welti, Michael; Troxler, Heinz; Hlsmeier, Andreas J; Imbach, Timo; Hennet, Thierry

    2011-02-25

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

  10. Improvement of Dolichol-linked Oligosaccharide Biosynthesis by the Squalene Synthase Inhibitor Zaragozic Acid*

    PubMed Central

    Haeuptle, Micha A.; Welti, Michael; Troxler, Heinz; Hlsmeier, Andreas J.; Imbach, Timo; Hennet, Thierry

    2011-01-01

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc2Man5 in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

  11. Glycogen Synthase Kinase-3 Inhibitors as Potent Therapeutic Agents for the Treatment of Parkinson Disease.

    PubMed Central

    2012-01-01

    Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of the nigrostriatal dopaminergic pathway. Because the current therapies only lead to temporary, limited improvement and have severe side effects, new approaches to treat PD need to be developed. To discover new targets for potential therapeutic intervention, a chemical genetic approach involving the use of small molecules as pharmacological tools has been implemented. First, a screening of an in-house chemical library on a well-established cellular model of PD was done followed by a detailed pharmacological analysis of the hits. Here, we report the results found for the small heterocyclic derivative called SC001, which after different enzymatic assays was revealed to be a new glycogen synthase kinase-3 (GSK-3) inhibitor with IC50 = 3.38 0.08 ?M. To confirm that GSK-3 could be a good target for PD, the evaluation of a set of structurally diverse GSK-3 inhibitors as neuroprotective agents for PD was performed. Results show that inhibitors of GSK-3 have neuroprotective effects in vitro representing a new pharmacological option for the disease-modifying treatment of PD. Furthermore, we show that SC001 is able to cross the bloodbrain barrier, protects dopaminergic neurons, and reduces microglia activation in in vivo models of Parkinson disease, being a good candidate for further drug development. PMID:23421686

  12. Inhibitors of nitric oxide synthase enhance rat ileum contractions induced by ricinoleic acid in vitro.

    PubMed

    Izzo, A A; Mascolo, N; Viola, P; Capasso, F

    1993-10-12

    The effects of NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, were studied on ricinoleic acid-evoked contractions in rat isolated ileum. Ricinoleic acid (10(-5) to 10(-4) M) caused a concentration-dependent contraction. Addition of L-NAME (30-300 microM) or L-NMMA (30-300 microM) to the Tyrode's solution increased in a concentration-dependent fashion the amplitude of the ricinoleic acid-evoked responses. L-Arginine (900 microM), a natural substrate of NO synthase, but not D-arginine (900 microM), counteracted the effect of L-NAME (300 microM). The potentiating effect of L-NAME was also prevented by sodium nitroprusside (0.1-1 microM), a generator of NO. These results provide evidence that endogenous NO may modulate the contraction of rat ileum induced by ricinoleic acid. As the contraction induced by ricinoleic acid is not blocked by tetrodotoxin (0.6 and 6.0 microM) the contractile effect of ricinoleic acid results mainly from a direct action on the smooth muscle. PMID:7504631

  13. Inhibitors of nitric oxide synthase enhance rat ileum contractions induced by ricinoleic acid in vitro.

    TOXLINE Toxicology Bibliographic Information

    Izzo AA; Mascolo N; Viola P; Capasso F

    1993-10-12

    The effects of NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, were studied on ricinoleic acid-evoked contractions in rat isolated ileum. Ricinoleic acid (10(-5) to 10(-4) M) caused a concentration-dependent contraction. Addition of L-NAME (30-300 microM) or L-NMMA (30-300 microM) to the Tyrode's solution increased in a concentration-dependent fashion the amplitude of the ricinoleic acid-evoked responses. L-Arginine (900 microM), a natural substrate of NO synthase, but not D-arginine (900 microM), counteracted the effect of L-NAME (300 microM). The potentiating effect of L-NAME was also prevented by sodium nitroprusside (0.1-1 microM), a generator of NO. These results provide evidence that endogenous NO may modulate the contraction of rat ileum induced by ricinoleic acid. As the contraction induced by ricinoleic acid is not blocked by tetrodotoxin (0.6 and 6.0 microM) the contractile effect of ricinoleic acid results mainly from a direct action on the smooth muscle.

  14. Tandem Benzophenone Amino Pyridines, Potent and Selective Inhibitors of Human Leukotriene C4 Synthase.

    PubMed

    Kleinschmidt, Thea K; Haraldsson, Martin; Basavarajappa, Devaraj; Lundeberg, Erik; Thulasingam, Madhuranayaki; Ekoff, Maria; Fauland, Alexander; Lehmann, Christoph; Kahnt, Astrid S; Lindbom, Lennart; Haeggstrm, Jesper Z

    2015-10-01

    Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C4 synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of three tandem benzophenone amino pyridines as inhibitors of LTC4S in vitro and in vivo. The inhibitors were characterized in vitro using recombinant human LTC4S, MonoMac 6 cells, and a panel of peripheral human immune cells. In vivo, the compounds were tested in the Zymosan A-induced peritonitis mouse model. The molecules, denoted TK04, TK04a, and TK05, were potent and selective inhibitors of LTC4S with IC50 values of 116, 124, and 95 nM, respectively. Molecular docking revealed binding in a hydrophobic crevice between two enzyme monomers and interaction with two catalytic residues, Arg104 and Arg31. The TK compounds potently inhibited cys-LT biosynthesis in immune cells. In coincubations of platelets and polymorphonuclear leukocytes, inhibition of LTC4S led to shunting of LTA4 toward anti-inflammatory lipoxin A4, which was significantly enhanced by simultaneous inhibition of LTA4H. Finally, we found that TK05 (6 mg?kg(-1)?body weight) reduces LTE4 levels in peritoneal lavage fluid by 88% and significantly decreases vascular permeability in vivo. Our findings indicate that the TK compounds are valuable experimental tools in eicosanoid research in vitro and in vivo. Their chemical structures may serve as leads for further inhibitor design. Novel drugs depleting cys-LT production could be beneficial for treatment of inflammatory diseases associated with overexpression of LTC4S. PMID:26283693

  15. Design, Synthesis, and Biochemical Evaluation of 1,5,6,7-Tetrahydro-6,7-dioxo-9-D-Ribitylaminolumazines Bearing Alkyl Phosphate Substituents as Inhibitors of Lumazine Synthase and Riboflavin Synthase

    PubMed Central

    Cushman, Mark; Jin, Guangyi; Illarionov, Boris; Fischer, Markus; Ladenstein, Rudolf; Bacher, Adelbert

    2008-01-01

    The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. In order to obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analog inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with Ki values in the low nanomolar range. The additional carbonyl in the dioxolumazine system vs. the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase. PMID:16277343

  16. Design, synthesis, and biochemical evaluation of 1,5,6,7-tetrahydro-6,7-dioxo-9-D-ribitylaminolumazines bearing alkyl phosphate substituents as inhibitors of lumazine synthase and riboflavin synthase.

    PubMed

    Cushman, Mark; Jin, Guangyi; Sambaiah, Thota; Illarionov, Boris; Fischer, Markus; Ladenstein, Rudolf; Bacher, Adelbert

    2005-09-30

    The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. To obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of Bacillus subtilislumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with Ki values in the low nanomolar range. The additional carbonyl in the dioxolumazine system versus the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase. PMID:16277343

  17. Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

    PubMed

    Lim, Won-Mook; Baig, Irshad Jameel; La, Im Joung; Choi, Jung-Do; Kim, Dong-Eun; Kim, Sung-Kun; Hyun, Jae-Wook; Kim, Giyoung; Kang, Chang-Ho; Kim, Young Jin; Yoon, Moon-Young

    2011-12-01

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds. PMID:22015678

  18. Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

    PubMed Central

    Jung, Carla S.; Wispel, Christian; Zweckberger, Klaus; Beynon, Christopher; Hertle, Daniel; Sakowitz, Oliver W.; Unterberg, Andreas W.

    2014-01-01

    Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment. PMID:24663083

  19. Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

    PubMed Central

    Noble, Brenda L.; Mendez, Daniel C.; Caseley, Paul S.; Peterson, Sarah C.; Routledge, Tyler J.; Patel, Nilay V.

    2012-01-01

    Canonical Wnt/?-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months. PMID:22745733

  20. Syntheses and herbicidal activity of new triazolopyrimidine-2-sulfonamides as acetohydroxyacid synthase inhibitor.

    PubMed

    Chen, Chao-Nan; Chen, Qiong; Liu, Yu-Chao; Zhu, Xiao-Lei; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

    2010-07-15

    The triazolopyrimidine-2-sulfonanilide, discovered from preparing bioisosteres of the sulfonylurea herbicides, is an important class of acetohydroxyacid synthase (AHAS, EC 4.1.3.18) inhibitors. At least over ten triazolopyrimidine sulfonanilides have been commercialized as herbicides for the control of broadleaf weeds and grass with cereal crop selectivity. Herein, a series of triazolopyrimidine-2-sulfonanilides were designed and synthesized with the aim of discovery of new herbicides with higher activity. The assay results of the inhibition activity of the synthesized compounds against Arabidopsis thatiana AHAS indicated that some compounds showed a little higher activity against flumetsulam (FS), the first commercial triazolopyrimidine-2-sulfonanilide-type herbicide. The ki values of two promising compounds 3d and 8h are respectively, 1.61 and 1.29 microM, while that of FS is 1.85 microM. Computational simulation results indicated the ester group of compound 3d formed hydrogen bonds with the surrounding residues Arg'198 and Ser653, which accounts for its 11.5-folds higher AHAS inhibition activity than Y6610. Further green house assay showed that compound 3d has comparable herbicidal activity as FS. Even at the concentration of 37.5g.ai/ha, 3d showed excellent herbicidal activity against Galium aparine, Cerastium arvense, Chenopodium album, Amaranthus retroflexus, and Rmumex acetasa, moderate herbicidal activity against Polygonum humifusum, Cyperus iria, and Eclipta prostrate. The combination of in vitro and in vivo assay indicated that 3d could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study. PMID:20598554

  1. Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase.

    PubMed

    Cho, June-Haeng; Lee, Mi-Young; Baig, Irshad Ahmed; Ha, Na-Reum; Kim, Joungmok; Yoon, Moon-Young

    2013-07-01

    Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors. PMID:23523771

  2. Unexpected Binding Modes of Nitric Oxide Synthase Inhibitors Effective in the Prevention of Cerebral Palsy Phenotype in an Animal Model

    PubMed Central

    Delker, Silvia L; Ji, Haitao; Li, Huiying; Jamal, Joumana; Fang, Jianguo; Xue, Fengtian; Silverman, Richard B.; Poulos, Thomas L.

    2011-01-01

    Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However, given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS inhibitors exhibit the highest level of selectivity over eNOS reported so far (? 3,800-fold). By using a combination of crystallography, computational methods, and site-directed mutagenesis, we found that inhibitor chirality and an unanticipated structural change of the target enzyme control both the orientation and selectivity of these novel nNOS inhibitors. A new hot spot generated owing to enzyme elasticity provides important information for the future fragment-based design of selective NOS inhibitors. PMID:20337441

  3. Area Wide Field Study on Effect of Three Chitin Synthesis Inhibitor Baits on Populations of Coptotermes formosanus Shiraki and Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Periodic sampling of 43 independent monitors, initially active with Formosan subterranean termite, Coptotermes formosanus Shiraki, or the eastern subterranean termite, Reticulitermes flavipes (Kollar) was conducted to evaluate the effects of cellulose baits containing one of three chitin synthesis i...

  4. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

    PubMed Central

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  5. Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

    PubMed

    Jones, T R; Varney, M D; Webber, S E; Lewis, K K; Marzoni, G P; Palmer, C L; Kathardekar, V; Welsh, K M; Webber, S; Matthews, D A; Appelt, K; Smith, W W; Janson, C A; Villafranca, J E; Bacquet, R J; Howland, E F; Booth, C L; Herrmann, S M; Ward, R W; White, J; Moomaw, E W; Bartlett, C A; Morse, C A

    1996-02-16

    To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration. PMID:8632414

  6. Glucosylceramide synthase inhibitors sensitise CLL cells to cytotoxic agents without reversing P-gp functional activity.

    PubMed

    Gerrard, Gareth; Butters, Terry D; Ganeshaguru, Kanagasabai; Mehta, Atul B

    2009-05-01

    Malignant B-cells from most chronic lymphocytic leukaemia (CLL) patients over-express MDR1 encoded P-glycoprotein (P-gp) multidrug efflux pump. Inhibition of glucosylceramide (GC) synthesis has been shown in cell lines to correlate with the expression and function of P-gp and sensitise cancer cells to cytotoxic agents. We investigated the hypothesis that reducing intracellular GC levels will reduce P-gp expression in malignant cells from CLL patients. We studied the ability of glucosylceramide synthase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1) and N-nonyl-deoxygalactonojirimycin (OGB-2) to sensitise CLL cells to conventional cytotoxic drug 2-chlorodeoxyadenosine (CdA) and the cytostatic drugs chlorambucil and fludarabine. The effect on P-gp activity was analysed using the calcein-AM accumulation assay where a multidrug activity factor (MAF) of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. The P-gp over-expressing cell line CEM-VLB showed a MAF value of 96.4 with the P-gp inhibitor Z.3HCL, which fell to 15.7 after co-incubation with OGB-1 and 45.9 with OGB-2. The IC(50) for vincristine fell from >10 microg/ml to 55.5 ng/ml in the presence of OGB-2. In P-gp(+ve) peripheral blood mononuclear cells from three normal volunteers, the mean MAF values for Z.3HCL, OGB-1 and OGB-2 were 23.86, 1.83 and 16.2 respectively. In 9/13 CLL samples the mean P-gp functional activity was 22.15 and P-gp was over-expressed in 12/13 samples. However, the MAF value with OGB-1 and OGB-2 was <10. Nevertheless, sensitisation in CLL cells was observed by a reduction in the IC(50) in the presence of OGB-1 and OGB-2 with the conventional drugs. We conclude that although GCS inhibitors sensitize CLL cells to cytotoxic and cytostatic drugs, they do not appear to have any effect on P-gp functional activity. PMID:19285492

  7. Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase-3?

    PubMed Central

    Whittle, Brendan J R; Varga, Csaba; Psa, Anik; Molnr, Andor; Collin, Marika; Thiemermann, Christoph

    2005-01-01

    The effects of the inhibitors of glycogen synthase kinase-3? (GSK-3?), TDZD-8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat. Administration of the GSK-3? inhibitor TDZD-8 (0.1, 0.33 or 1.0?mg?kg?1, s.c., b.i.d., for 3 days) caused a dose-dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 010 scale. Likewise, following administration of the GSK-3? inhibitor SB 415286 (0.1, 0.33 or 1.0?mg?kg?1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS-provoked colonic inflammation was reduced. Administration of either TDZD-8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied. The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS-induced inflamed colon, was significantly inhibited by both TDZD-8 and SB 415286 at each dose level. The increase in the levels of the proinflammatory cytokine, TNF-?, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated. The elevated levels of the transcription factor NF-?B subunit p65, as determined by Western blot in the nuclear extracts from the TNBS-provoked inflamed colonic tissue, were dose-dependently reduced by TDZD-8 or SB 415286 treatment. These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK-3? reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti-inflammatory action may be related to downregulation of NF-?B activity, involved in the generation of proinflammatory mediators. PMID:16314851

  8. Glucose-modulated mitochondria adaptation in tumor cells: a focus on ATP synthase and inhibitor Factor 1.

    PubMed

    Domenis, Rossana; Bisetto, Elena; Rossi, Davide; Comelli, Marina; Mavelli, Irene

    2012-01-01

    Warburg's hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking "positive" (activation/biogenesis) or "negative" (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase ? subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-? suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on ?-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation. PMID:22408432

  9. Identification of a Glycogen Synthase Kinase-3? Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    PubMed Central

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-01-01

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called mood-stabilizing drugs, such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3? (GSK-3?) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3?. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC50 values in the range of 4 to 680 nm against human GSK-3?. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mgkg?1 resulted in the attenuation of hyperactivity in amphetamine/ chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mgkg?1) and the antipsychotic haloperidol (1 mgkg?1). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3? in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3? as a relevant therapeutic target in the identification of new therapies for bipolar patients. PMID:21732538

  10. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    SciTech Connect

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  11. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418.

    PubMed

    Bhat, Ratan; Xue, Yafeng; Berg, Stefan; Hellberg, Sven; Ormö, Mats; Nilsson, Yvonne; Radesäter, Ann-Cathrin; Jerning, Eva; Markgren, Per-Olof; Borgegård, Thomas; Nylöf, Martin; Giménez-Cassina, Alfredo; Hernández, Félix; Lucas, Jose J; Díaz-Nido, Javier; Avila, Jesús

    2003-11-14

    Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 +/- 27 nM), in an ATP-competitive manner (Ki = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 microM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3beta protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by beta-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5. PMID:12928438

  12. Effects of Sublethal Concentrations of the Chitin Synthesis Inhibitor, Hexaflumuron, on the Development and Hemolymph Physiology of the Cutworm, Spodoptera litura

    PubMed Central

    Zhu, Qiqi; He, Yuan; Yao, Jing; Liu, Yinzhao; Tao, Liming; Huang, Qingchun

    2012-01-01

    The effects of sublethal concentrations 0.1, 0.5, and 1.2 µg mL-1of the chitin synthesis inhibitor, hexaflumuron, on larval growth and development, the count and proportion of hemocytes, and carbohydrate content (trehalose and glyceride) in hemolymph were investigated in the cutworm, Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae). When 3rdinstar larvae were subjected to the sublethal concentrations, there were dose-dependent effects on larval weight and length of each instar larvae, percent pupation and the duration of development. Most of the larvae died during the molting process at all concentrations. Few individuals from 0.5 and 1.2 µg mL -1concentrations could develop to the 6thinstar, while the pupae emerging from the 0.1 µg mL -1concentrations did not exceed 16% of the number of the initial larvae. In 5thinstar S. litura, the total number of hemocytes was significantly increased at 24 hours post—treatment, whereas the proliferation of hemocytes was inhibited, plasmatocyte pseudopodia contracted, and granulocyte expanded at 96 hours post—treatment. The increases of plasmatocyte count and the decreases of granulocyte count were dose—dependent. The longer treatment time of the sublethal concentrations increased the content of total carbohydrate and trehalose in hematoplasma, and was dose—dependent in hemocytes. The content of glyceride in hemolymph was significantly higher at 24 hours post—treatment, but gradually returned to normal levels at 96 hours post—treatment as compared with the control. The results suggested that sublethal concentrations of hexaflumuron reduced S. litura larval survival and interfered with hemolymph physiological balances. PMID:22958164

  13. Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.).

    PubMed

    Liu, Weitang; Bi, Yaling; Li, Lingxu; Yuan, Guohui; Du, Long; Wang, Jinxin

    2013-09-01

    Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I50 value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene. PMID:25149235

  14. Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors.

    PubMed

    Choi, Kyoung-Jae; Noh, Kyoung Mi; Kim, Dong-Eun; Ha, Byung Hak; Kim, Eunice Eunkyung; Yoon, Moon-Young

    2007-10-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate- (ThDP)- and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids (BCAAs) leucine, isoleucine, and valine. The gene from Haemophilus influenzae that encodes the AHAS catalytic subunit was cloned, overexpressed in Escherichia coli BL21(DE3), and purified to homogeneity. The purified H. influenzae AHAS catalytic subunit (Hin-AHAS) appeared as a single band on SDS-PAGE gel, with a molecular mass of approximately 63 kDa. The enzyme catalyzes the condensation of two molecules of pyruvate to form acetolactate, with a K(m) of 9.2mM and the specific activity of 1.5 micromol/min/mg. The cofactor activation constant (K(c)=13.5 microM) and the dissociation constant (K(d)=3.3 microM) of ThDP were also determined by enzymatic assay and tryptophan fluorescence quenching studies, respectively. We screened a chemical library to discover new inhibitors of the Hin AHAS catalytic subunit. Through which, AVS-2087 (IC(50)=0.53 microM), KSW30191 (IC(50)=1.42 microM), and KHG20612 (IC(50)=4.91 microM) displayed potent inhibition as compare to sulfometuron methyl (IC(50)=276.31 microM). PMID:17718999

  15. Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.

    PubMed

    Lee, Yu-Ting; Cui, Chang-Jun; Chow, Eve W L; Pue, Nason; Lonhienne, Thierry; Wang, Jian-Guo; Fraser, James A; Guddat, Luke W

    2013-01-10

    The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell. PMID:23237384

  16. Pharmacodynamic comparison of LY3023703, a novel microsomal prostaglandin e synthase 1 inhibitor, with celecoxib.

    PubMed

    Jin, Y; Smith, C L; Hu, L; Campanale, K M; Stoltz, R; Huffman, L G; McNearney, T A; Yang, X Y; Ackermann, B L; Dean, R; Regev, A; Landschulz, W

    2016-03-01

    To assess the safety, tolerability, and pharmacology of LY3023703, a microsomal prostaglandin E synthase 1 (mPGES1) inhibitor, a multiple ascending dose study was conducted. Forty-eight subjects received LY3023703, celecoxib (400 mg), or placebo once daily for 28 days. Compared with placebo, LY3023703 inhibited ex vivo lipopolysaccharide-stimulated prostaglandin E2 (PGE2 ) synthesis 91% and 97% on days 1 and 28, respectively, after 30-mg dosing, comparable to celecoxib's effect (82% inhibition compared to placebo). Unlike celecoxib, which also inhibited prostacyclin synthesis by 44%, LY3023703 demonstrated a maximal increase in prostacyclin synthesis of 115%. Transient elevations of serum aminotransferase were observed in one subject after 30-mg LY3023703 dosing (10 upper limit of normal (ULN)), and one subject after 15-mg dosing (about 1.5 ULN). Results from this study suggest that mPGES1 inhibits inducible PGE synthesis without suppressing prostacyclin generation and presents a novel target for inflammatory pain. PMID:26351780

  17. Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain.

    PubMed

    Larsen, Scott D; Wilson, Michael W; Abe, Akira; Shu, Liming; George, Christopher H; Kirchhoff, Paul; Showalter, H D Hollis; Xiang, Jianming; Keep, Richard F; Shayman, James A

    2012-02-01

    Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate. PMID:22058426

  18. Thiazolopyrimidine inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum.

    PubMed

    Geist, Julie G; Lauw, Susan; Illarionova, Victoria; Illarionov, Boris; Fischer, Markus; Grwert, Tobias; Rohdich, Felix; Eisenreich, Wolfgang; Kaiser, Johannes; Groll, Michael; Scheurer, Christian; Wittlin, Sergio; Alonso-Gmez, Jos L; Schweizer, W Bernd; Bacher, Adelbert; Diederich, Franois

    2010-07-01

    A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay. PMID:20480490

  19. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis.

    PubMed

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C

    2015-07-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

  20. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation

    PubMed Central

    Jin, Shuangxia; Singh, Nameirakpam D.; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-01-01

    Summary In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. PMID:25782349

  1. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation.

    PubMed

    Jin, Shuangxia; Singh, Nameirakpam D; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-04-01

    In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. PMID:25782349

  2. A new series of N-[2,4-dioxo-6-d-ribitylamino-1,2,3,4-tetrahydropyrimidin-5-yl]oxalamic acid derivatives as inhibitors of lumazine synthase and riboflavin synthase: design, synthesis, biochemical evaluation, crystallography, and mechanistic implications.

    PubMed

    Zhang, Yanlei; Illarionov, Boris; Morgunova, Ekaterina; Jin, Guangyi; Bacher, Adelbert; Fischer, Markus; Ladenstein, Rudolf; Cushman, Mark

    2008-04-01

    The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure. PMID:18331058

  3. Structural and thermodynamic insights into the binding mode of five novel inhibitors of lumazine synthase from Mycobacterium tuberculosis.

    PubMed

    Morgunova, Ekaterina; Illarionov, Boris; Sambaiah, Thota; Haase, Ilka; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2006-10-01

    Recently published genomic investigations of the human pathogen Mycobacterium tuberculosis have revealed that genes coding the proteins involved in riboflavin biosynthesis are essential for the growth of the organism. Because the enzymes involved in cofactor biosynthesis pathways are not present in humans, they appear to be promising candidates for the development of therapeutic drugs. The substituted purinetrione compounds have demonstrated high affinity and specificity to lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis in bacteria and plants. The structure of M. tuberculosis lumazine synthase in complex with five different inhibitor compounds is presented, together with studies of the binding reactions by isothermal titration calorimetry. The inhibitors showed the association constants in the micromolar range. The analysis of the structures demonstrated the specific features of the binding of different inhibitors. The comparison of the structures and binding modes of five different inhibitors allows us to propose the ribitylpurinetrione compounds with C4-C5 alkylphosphate chains as most promising leads for further development of therapeutic drugs against M. tuberculosis. PMID:16984393

  4. Heme-Coordinating Inhibitors of Neuronal Nitric Oxide Synthase. Iron-Thioether Coordination is Stabilized by Hydrophobic Contacts Without Increased Inhibitor Potency

    PubMed Central

    Martell, Jeffrey D.; Li, Huiying; Doukov, Tzanko; Martásek, Pavel; Roman, Linda J.; Soltis, Michael; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge, the first crystal structure of a thioether inhibitor complexed to any heme enzyme. A series of related potential inhibitors (4-8) also were evaluated. Compounds 4-8 were all found to be type I (non-heme-coordinating) inhibitors of ferric nNOS, but 4 and 6-8 were found to switch to type II upon heme reduction to the ferrous state, reflecting the higher affinity of thioethers for ferrous heme than for ferric heme. Contrary to what has been widely thought, thioether-heme ligation was found not to increase inhibitor potency, illustrating the intrinsic weakness of the thioether-ferric heme linkage. Subtle changes in the alkyl groups attached to the thioether sulfur caused drastic changes in binding conformation, indicating that hydrophobic contacts play a crucial role in stabilizing the thioether-heme coordination. PMID:20014790

  5. Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

    PubMed

    Baig, Irshad Ahmed; Moon, Ji-Young; Lee, Sang-Choon; Ryoo, Sung-Weon; Yoon, Moon-Young

    2015-10-01

    Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 μg/ml and 6.24 μg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents. PMID:25988243

  6. Docking and 3D-QSAR studies of acetohydroxy acid synthase inhibitor sulfonylurea derivatives.

    PubMed

    Roy, Kunal; Paul, Somnath

    2010-05-01

    Docking and three dimensional quantitative-structure activity relationship (3D-QSAR) studies were performed on acetohydroxy acid synthase (AHAS) inhibitor sulfonylurea analogues with potential herbicidal activity. The 3D-QSAR studies were carried out using shape, spatial and electronic descriptors along with a few structural parameters. Genetic function approximation (GFA) was used as the chemometric tool for this analysis. The whole data set (n = 45) was divided into a training set (75% of the data set) and a test set (remaining 25%) on the basis of the K-means clustering technique on a standardised topological, physicochemical and structural descriptor matrix. Models developed from the training set were used to predict the activity of the test set compounds. All models were validated internally, externally and using the Y-randomisation technique. Docking studies suggested that the molecules bind within a pocket of the enzyme formed by some important amino acid residues (Met351, Asp375, Arg377, Gly509, Met570 and Val571). In QSAR studies, molecular shape analysis showed that bulky substitution at the R(1) position may enhance AHAS inhibitory activity. Charged surface area descriptors suggested that negative charge distributed over a large surface area may enhance this activity. The hydrogen bond acceptor parameter supported the charged surface area descriptors and suggested that, for better activity, the number of electronegative atoms present in the molecule should be high. The spatial descriptors show that, for better activity, the molecules should possess a bulky substituent and a small substitution at the R(2) and R(3) positions, respectively. PMID:19841951

  7. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro.

    PubMed Central

    Papapetropoulos, A.; Desai, K. M.; Rudic, R. D.; Mayer, B.; Zhang, R.; Ruiz-Torres, M. P.; Garca-Cardea, G.; Madri, J. A.; Sessa, W. C.

    1997-01-01

    Angiogenesis is a complex process involving endothelial cell (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-beta 1. Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-beta 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs. Images Figure 2 Figure 4 Figure 5 PMID:9137106

  8. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    PubMed

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death. PMID:18603020

  9. Discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as riboflavin synthase inhibitors with antibiotic activity against Mycobacterium tuberculosis.

    PubMed

    Zhao, Yujie; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Georg, Gunda; Ye, Qi-Zhuang; Fanwick, Phillip E; Franzblau, Scott G; Wan, Baojie; Cushman, Mark

    2009-08-01

    A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli riboflavin synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a k(is) of 8.7 microM vs. M. tuberculosis riboflavin synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 microM vs. M. tuberculosis replicating phenotype and 48.9 microM vs. M. tuberculosis nonreplicating phenotype. The HTS hit compound and its analogues provide the first examples of riboflavin synthase inhibitors with antibiotic activity. PMID:19545132

  10. Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase.

    PubMed

    Lobo, A P; Nair, M G; Changchien, L; Weichsel, A; Montfort, W R; Maley, F

    1998-03-31

    5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS. PMID:9521774

  11. Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites

    PubMed Central

    Mori, Mihoko; Jeelani, Ghulam; Masuda, Yui; Sakai, Kazunari; Tsukui, Kumiko; Waluyo, Danang; Tarwadi; Watanabe, Yoshio; Nonaka, Kenichi; Matsumoto, Atsuko; Ōmura, Satoshi; Nozaki, Tomoyoshi; Shiomi, Kazuro

    2015-01-01

    Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to CS inhibition. PMID:26441896

  12. Bacterial acetohydroxyacid synthase and its inhibitors--a summary of their structure, biological activity and current status.

    PubMed

    Gedi, Vinayakumar; Yoon, Moon-Young

    2012-03-01

    Acetohydroxyacid synthase (anabolic AHAS; EC 2.2.1.6) is a thiamin diphosphate-dependent enzyme that catalyzes the first step in the branched-chain amino acid (BCAA) biosynthesis pathway. BCAAs are synthesized by plants, algae, fungi and bacteria, although not by animals. Thus, the enzymes of the BCAA biosynthetic pathway are potential targets in the development of herbicides, fungicides and antimicrobial compounds. Plant AHASs are well studied in this regard because specific plant AHAS inhibitors are considered to comprise the most potent herbicides. These inhibitors are also effective against bacterial AHASs, inhibit the growth of several bacterial strains and have little to no toxicity in mammals. This review provides an overview of bacterial AHASs with an update of the current status of AHAS inhibitors. PMID:22284339

  13. Structure-guided Discovery of Phenyl diketo-acids as Potent Inhibitors of M. tuberculosis Malate Synthase

    PubMed Central

    Krieger, Inna V.; Freundlich, Joel S.; Gawandi, Vijay B.; Roberts, Justin P.; Gawandi, Vidyadhar B.; Sun, Qingan; Owen, Joshua L.; Fraile, Maria T.; Huss, Sofia I.; Lavandera, Jose-Luis; Ioerger, Thomas R.; Sacchettini, James C.

    2012-01-01

    Summary The glyoxylate shunt plays an important role in fatty-acid metabolism, and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of novel phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and over-expression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors were used to guide optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics. PMID:23261599

  14. Cell Wall Polysaccharide Synthases Are Located in Detergent-Resistant Membrane Microdomains in Oomycetes ?

    PubMed Central

    Briolay, Anne; Bouzenzana, Jamel; Guichardant, Michel; Deshayes, Christian; Sindt, Nicolas; Bessueille, Laurence; Bulone, Vincent

    2009-01-01

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1?3)-?-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1?3)-?-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth. PMID:19201970

  15. Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation

    SciTech Connect

    Zhang, Y.; Cao, R; Yin, F; Hudock, M; Guo, R; Song, Y; No, J; Bergan, K; Leon, A; et al,

    2009-01-01

    Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

  16. Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in Fragile X mice

    PubMed Central

    Franklin, Aimee V.; King, Margaret K.; Palomo, Valle; Martinez, Ana; McMahon, Lori L.; Jope, Richard S.

    2013-01-01

    Background Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of Fragile X Syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes. Methods We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor (NMDAR)-dependent long term potentiation (LTP) at medial perforant path synapses onto dentate granule cells (MPP-DGC) and dentate gyrus-dependent cognitive behavioral tasks. Results GSK3 inhibitors completely rescued deficits in LTP at MPP-DGC synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent congnitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects. Conclusions These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS. PMID:24041505

  17. Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase

    PubMed Central

    Lidor, O.; Al-Quntar, A.; Pesci, E. C.; Steinberg, D.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases. PMID:26593271

  18. Biological adhesive based on carboxymethyl chitin derivatives and chitin nanofibers.

    PubMed

    Azuma, Kazuo; Nishihara, Masahiro; Shimizu, Haruki; Itoh, Yoshiki; Takashima, Osamu; Osaki, Tomohiro; Itoh, Norihiko; Imagawa, Tomohiro; Murahata, Yusuke; Tsuka, Takeshi; Izawa, Hironori; Ifuku, Shinsuke; Minami, Saburo; Saimoto, Hiroyuki; Okamoto, Yoshiharu; Morimoto, Minoru

    2015-02-01

    Novel biological adhesives made from chitin derivatives were prepared and evaluated for their adhesive properties and biocompatibility. Chitin derivatives with acrylic groups, such as 2-hydroxy-3-methacryloyloxypropylated carboxymethyl chitin (HMA-CM-chitin), were synthesized and cured by the addition of an aqueous hydrogen peroxide solution as a radical initiator. The adhesive strength of HMA-CM-chitin increased when it was blended with chitin nanofibers (CNFs) or surface-deacetylated chitin nanofibers (S-DACNFs). HMA-CM-chitin/CNFs or HMA-CM-chitin/S-DACNFs have almost equal adhesive strength compared to that of a commercial cyanoacrylate adhesive. Moreover, quick adhesion and induction of inflammatory cells migration were observed in HMA-CM-chitin/CNF and HMA-CM-chitin/S-DACNF. These findings indicate that the composites prepared in this study are promising materials as new biological adhesives. PMID:25542790

  19. Temperature dependent spin crossover in neuronal nitric oxide synthase bound with the heme-coordinating thioether inhibitors

    PubMed Central

    Doukov, Tzanko; Li, Huiying; Sharma, Ajay; Martell, Jeffrey D.; Soltis, Michael; Silverman, Richard B.; Poulos, Thomas L.

    2011-01-01

    A series of L-arginine analogue nitric oxide synthase inhibitors with a thioether tail have been shown to form an Fe-S thioether interaction as evidenced by continuous electron density between the Fe and S atoms. Even so, the Fe-S thioether interaction was found to be far less important for inhibitor binding than the hydrophobic interactions between the alkyl group in the thioether tail and surrounding protein (Martell et al., (2010) J. Am. Chem. Soc. 132, 798). However, among the few thioether inhibitors that showed Fe-S thioether interaction in crystal structures, variations in spin state (high-spin or low-spin) were observed dependent upon the heme iron oxidation state and temperature. Since modern synchrotron X-ray data collection is typically carried out at cryogenic temperatures, we reasoned that some of the discrepancies between cryo-crystal structures and room temperature UV-visible spectroscopy could be the result of temperature dependent spin-state changes. We, therefore, have characterized some of these nNOS-thioether inhibitor complexes in both crystal and solution using EPR and UV-visible absorption spectrometry as a function of temperature and the heme iron redox state. We found that some thioether inhibitors switch from high- to low-spin at lower temperatures similar to the “spin crossover” phenomenon observed in many transition metal complexes. PMID:21534614

  20. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2014-06-01

    Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families. PMID:23627736

  1. Discovery of novel acetohydroxyacid synthase inhibitors as active agents against Mycobacterium tuberculosis by virtual screening and bioassay.

    PubMed

    Wang, Di; Zhu, Xuelian; Cui, Changjun; Dong, Mei; Jiang, Hualiang; Li, Zhengming; Liu, Zhen; Zhu, Weiliang; Wang, Jian-Guo

    2013-02-25

    Acetohydroxyacid synthase (AHAS) has been regarded as a promising drug target against Mycobacterium tuberculosis (MTB) as it catalyzes the biosynthesis of branched-chain amino acids. In this study, 23 novel AHAS inhibitors were identified through molecular docking followed by similarity search. The determined IC(50) values range from 0.385 ± 0.026 μM to >200 μM against bacterium AHAS. Five of the identified compounds show significant in vitro activity against H37Rv strains (MICs in the range of 2.5-80 mg/L) and clinical MTB strains, including MDR and XDR isolates. More impressively, compounds 5 and 7 can enhance the killing ability against macrophages infected pathogen remarkably. This study suggests our discovered inhibitors can be further developed as novel anti-MTB therapeutics targeting AHAS. PMID:23316686

  2. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    SciTech Connect

    Sharma, Bhupesh Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in As induced VaD.

  3. L-NAME, a nitric oxide synthase inhibitor, as a potential countermeasure to post-suspension hypotension in rats

    NASA Technical Reports Server (NTRS)

    Bayorh, M. A.; Socci, R. R.; Watts, S.; Wang, M.; Eatman, D.; Emmett, N.; Thierry-Palmer, M.

    2001-01-01

    A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30 degrees tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

  4. Novel inhibitors of the condensing enzymes of the type II fatty acid synthase of pea (Pisum sativum).

    PubMed Central

    Jones, A L; Herbert, D; Rutter, A J; Dancer, J E; Harwood, J L

    2000-01-01

    The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called beta-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-(14)C]acetate substrate and chloroplast preparations from pea leaves, and [1-(14)C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens. PMID:10727420

  5. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs

    SciTech Connect

    Nishimoto, Tomoyuki; Ishikawa, Eiichiro; Anayama, Hisashi; Hamajyo, Hitomi; Nagai, Hirofumi; Hirakata, Masao; Tozawa, Ryuichi

    2007-08-15

    High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy.

  6. First report on chitinous holdfast in sponges (Porifera)

    PubMed Central

    Ehrlich, Hermann; Kaluzhnaya, Oksana V.; Tsurkan, Mikhail V.; Ereskovsky, Alexander; Tabachnick, Konstantin R.; Ilan, Micha; Stelling, Allison; Galli, Roberta; Petrova, Olga V.; Nekipelov, Serguei V.; Sivkov, Victor N.; Vyalikh, Denis; Born, Ren; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I.; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V.; Wrheide, Gert

    2013-01-01

    A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, MorganElson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to ?-chitin than to ?-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates. PMID:23677340

  7. First report on chitinous holdfast in sponges (Porifera).

    PubMed

    Ehrlich, Hermann; Kaluzhnaya, Oksana V; Tsurkan, Mikhail V; Ereskovsky, Alexander; Tabachnick, Konstantin R; Ilan, Micha; Stelling, Allison; Galli, Roberta; Petrova, Olga V; Nekipelov, Serguei V; Sivkov, Victor N; Vyalikh, Denis; Born, Ren; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V; Wrheide, Gert

    2013-07-01

    A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to ?-chitin than to ?-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates. PMID:23677340

  8. Novel 2,4-Disubstituted Pyrimidines as Potent, Selective, and Cell-Permeable Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    2014-01-01

    Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes. PMID:25489882

  9. Novel 2,4-disubstituted pyrimidines as potent, selective, and cell-permeable inhibitors of neuronal nitric oxide synthase.

    PubMed

    Mukherjee, Paramita; Li, Huiying; Sevrioukova, Irina; Chreifi, Georges; Martásek, Pavel; Roman, Linda J; Poulos, Thomas L; Silverman, Richard B

    2015-02-12

    Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure-activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes. PMID:25489882

  10. The current state of resistance to acetohydroxyacid/acetolactate synthase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The acetohydroxyacid/acetolactate synthase (ALS) inhibiting herbicides are used for weed management in multiple crop and non-crop situations. Herbicides with this mechanism of action were introduced in the early 1980s and quickly came to dominate many cropping situations due to their broad spectrum...

  11. Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation

    PubMed Central

    Selenica, M-L; Jensen, H S; Larsen, A K; Pedersen, M L; Helboe, L; Leist, M; Lotharius, J

    2007-01-01

    Background and purpose: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3β expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3β inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3β enzyme activity levels in 12-day old postnatal rats. Experimental approach: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser396) levels in brain tissue was measured by immunoblotting and correlated with GSK-3β enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3β activities and cell death were measured in a human neuronal cell line (LUHMES). Key results: Lithium and CHIR98014 reduced tau phosphorylation (Ser396) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3′-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC50 values obtained in recombinant or cell-based GSK-3β enzyme activity assays. The inhibitory effect on GSK-3β activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low μM inhibitor concentrations. Conclusions and Implications: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3β may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis. PMID:17906685

  12. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

    PubMed Central

    Salvemini, D; Manning, P T; Zweifel, B S; Seibert, K; Connor, J; Currie, M G; Needleman, P; Masferrer, J L

    1995-01-01

    We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced. Images PMID:7542281

  13. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    SciTech Connect

    Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-03-01

    In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

  14. [Effects of dimilin, a chitin inhibitor 1 (4 chlorophenyl) 3 (2-6 difluorobenzoyl) urea on the oenocytes and molting in the processionary caterpillar (Thaumetopoea pityocampa Schiff.) (Lepidoptera) (author's transl)].

    PubMed

    Denneulin, J C; Lamy, M

    1977-01-01

    Te oenocytes of the processionary caterpillar show histophysiological variations during their developing cycle. Grafting experiments and culture in vitro, have not so far allowed us to reveal the least participation of the oenocytes in the determinism of molting and in the transformation of cholesterol into ecdysone. On the other hand, histochemical studies of the oenocytes during the last period of their larval state, reveal, just before nymphosis, the existence of polysaccharides which probably correspond to the synthesis of pre-cuticular substance. When the caterpillars are treated with a chitin inhibitor (pH - 60-40 = Dimilin), the polysaccharides are not longer to be seen in the oenocytes. This deficiency in cuticular material could well be the consequence of one of the most spectacular effects of this product that is a profound perturbation in the formation of the cuticle that leads to the death of animals when molting. PMID:565611

  15. The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases.

    PubMed

    Li, Huiying; Jamal, Joumana; Delker, Silvia; Plaza, Carla; Ji, Haitao; Jing, Qing; Huang, He; Kang, Soosung; Silverman, Richard B; Poulos, Thomas L

    2014-08-19

    Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity. PMID:25089924

  16. The Mobility of a Conserved Tyrosine Residue Controls Isoform-Dependent Enzyme–Inhibitor Interactions in Nitric Oxide Synthases

    PubMed Central

    2015-01-01

    Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS–inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity. PMID:25089924

  17. The occurrence of chitin in the hemocytes of invertebrates

    PubMed Central

    Heath-Heckman, Elizabeth A.C.; McFall-Ngai, Margaret J.

    2011-01-01

    The light-organ symbiosis of Euprymna scolopes, the Hawaiian bobtail squid, is a useful model for the study of animal–microbe interactions. Recent analyses have demonstrated that chitin breakdown products play a role in communication between E. scolopes and its bacterial symbiont Vibrio fischeri. In this study, we sought to determine the source of chitin in the symbiotic organ. We used a commercially available chitin-binding protein (CBP) conjugated to fluorescein to label the polymeric chitin in host tissues. Confocal microscopy revealed that the only cells in contact with the symbionts that labeled with the probe were the macrophage-like hemocytes, which traffic into the light-organ crypts where the bacteria reside. Labeling of extracted hemocytes by CBP was markedly decreased following treatment with purified chitinase, providing further evidence that the labeled molecule is polymeric chitin. Further, CBP-positive areas co-localized with both a halide peroxidase antibody and Lysotracker, a lysosomal marker, suggesting that the chitin-like biomolecule occurs in the lysosome or acidic vacuoles. Reverse transcriptase polymerase chain reaction (PCR) of hemocytes revealed mRNA coding for a chitin synthase, suggesting that the hemocytes synthesize chitin de novo. Finally, upon surveying blood cells from other invertebrate species, we observed CBP-positive regions in all granular blood cells examined, suggesting that this feature is a shared character among the invertebrates; the vertebrate blood cells that we sampled did not label with CBP. Although the function of the chitin-like material remains undetermined, its presence and subcellular location in invertebrate hemocytes suggests a conserved role for this polysaccharide in the immune system of diverse animals. PMID:21723107

  18. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    PubMed Central

    Sprenger, Janina; Svensson, Bo; Hlander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-01-01

    The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5?-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. PMID:25760598

  19. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    PubMed

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, Jos A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having ?M antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. PMID:25900220

  20. Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.

    PubMed

    Ashe, Karen M; Bangari, Dinesh; Li, Lingyun; Cabrera-Salazar, Mario A; Bercury, Scott D; Nietupski, Jennifer B; Cooper, Christopher G F; Aerts, Johannes M F G; Lee, Edward R; Copeland, Diane P; Cheng, Seng H; Scheule, Ronald K; Marshall, John

    2011-01-01

    The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects. PMID:21738789

  1. Overview of chitin metabolism enzymes in Manduca sexta: Identification, domain organization, phylogenetic analysis and gene expression.

    PubMed

    Tetreau, Guillaume; Cao, Xiaolong; Chen, Yun-Ru; Muthukrishnan, Subbaratnam; Jiang, Haobo; Blissard, Gary W; Kanost, Michael R; Wang, Ping

    2015-07-01

    Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes. PMID:25616108

  2. Flavin-Dependent Thymidylate Synthase as a Drug Target for Deadly Microbes: Mutational Study and a Strategy for Inhibitor Design.

    PubMed

    Mathews, Irimpan I

    2013-04-20

    The identification of flavin-dependent thymidylate synthase (FDTS) as an essential enzyme and its occurrence in several pathogenic microbes opens opportunities for using FDTS enzyme as an excellent target for new antimicrobial drug discovery. In contrast to the human thymidylate synthase enzyme that utilizes methylene-tetrahydrofolate (CH2H4 folate) for the conversion of dUMP to dTMP, the microbial enzymes utilize an additional non-covalently bound FAD molecule for the hydride transfer from NAD(P)H. The structural and mechanistic differences between the human and microbial enzymes present an attractive opportunity for the design of antimicrobial compounds specific for the pathogens. We have determined the crystal structure of FDTS enzyme in complex with the methyl donor, CH2H4 folate. We describe here the structure of a FDTS mutant and compare it with other FDTS complex structures, including a FDTS-CH2H4 folate complex. We identified a conformational change essential for substrate binding and propose a strategy for the design of FDTS specific inhibitors. PMID:24563811

  3. Design and structure-activity relationships of potent and selective inhibitors of undecaprenyl pyrophosphate synthase (UPPS): tetramic, tetronic acids and dihydropyridin-2-ones.

    PubMed

    Peukert, Stefan; Sun, Yingchuan; Zhang, Rui; Hurley, Brian; Sabio, Mike; Shen, Xiaoyu; Gray, Christen; Dzink-Fox, JoAnn; Tao, Jianshi; Cebula, Regina; Wattanasin, Sompong

    2008-03-15

    Based on a pharmacophore hypothesis substituted tetramic and tetronic acid 3-carboxamides as well as dihydropyridin-2-one-3-carboxamides were investigated as inhibitors of undecaprenyl pyrophosphate synthase (UPPS) for use as novel antimicrobial agents. Synthesis and structure-activity relationship patterns for this class of compounds are discussed. Selectivity data and antibacterial activities for selected compounds are provided. PMID:18295483

  4. 3,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase (nNOS) inhibitors.

    PubMed

    Annedi, Subhash C; Maddaford, Shawn P; Ramnauth, Jailall; Renton, Paul; Speed, Joanne; Rakhit, Suman; Andrews, John S; Porreca, Frank

    2012-03-01

    A series of 3,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). Various guanidine isosteric groups were explored at the 5-position of the indole ring, while keeping the basic amine side chain such as N-methylpiperidine ring, fixed at the 3-position of the indole ring. Compounds having 2-thiophene amidine and 2-furanyl amidine groups (7, 8, 10 and 12) showed increased activity for human neuronal NOS and good selectivity over endothelial and inducible NOS isoforms. Compound 8 was shown to reverse (10mg/kg, ip) thermal hyperalgesia in the L(5)/L(6) spinal nerve ligation (neuropathic pain) model and was devoid of any significant drug-drug interaction potential due to cytochrome P450 inhibition or cardiovascular liabilities associated with the inhibition of endothelial NOS. PMID:22318159

  5. A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D-ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: design, synthesis, and evaluation.

    PubMed

    Zhang, Yanlei; Jin, Guangyi; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Cushman, Mark

    2007-09-14

    Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly. PMID:17705537

  6. Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut.

    PubMed

    Kelkenberg, Marco; Odman-Naresh, Jothini; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2015-01-01

    In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut. PMID:25449129

  7. Marine natural products as inhibitors of cystathionine beta-synthase activity.

    PubMed

    Thorson, Megan K; Van Wagoner, Ryan M; Harper, Mary Kay; Ireland, Chris M; Majtan, Tomas; Kraus, Jan P; Barrios, Amy M

    2015-03-01

    A library consisting of characterized marine natural products as well as synthetic derivatives was screened for compounds capable of inhibiting the production of hydrogen sulfide (H2S) by cystathionine beta-synthase (CBS). Eight hits were validated and shown to inhibit CBS activity with IC50 values ranging from 83 to 187?M. The majority of hits came from a series of synthetic polyandrocarpamine derivatives. In addition, a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions is reported. PMID:25666819

  8. Fatty Acid Synthase Inhibitors Induce Apoptosis in Non-Tumorigenic Melan-A Cells Associated with Inhibition of Mitochondrial Respiration

    PubMed Central

    Rossato, Franco A.; Zecchin, Karina G.; La Guardia, Paolo G.; Ortega, Rose M.; Alberici, Luciane C.; Costa, Rute A. P.; Catharino, Rodrigo R.; Graner, Edgard; Castilho, Roger F.; Vercesi, Aníbal E.

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition. PMID:24964211

  9. Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

    PubMed

    Pham, Ngoc Chien; Moon, Ji-Young; Cho, Jun-Haeng; Lee, Soo-Jae; Park, Joon-Shik; Kim, Dong-Eun; Park, Yoonkyung; Yoon, Moon-Young

    2010-01-01

    The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds. PMID:21071847

  10. The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

    PubMed Central

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

  11. Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase.

    PubMed

    Cinelli, Maris A; Li, Huiying; Pensa, Anthony V; Kang, Soosung; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2015-11-12

    Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding. PMID:26469213

  12. Pharmacophore Modeling and Virtual Screening for Novel Acidic Inhibitors of Microsomal Prostaglandin E2 Synthase-1 (mPGES-1)

    PubMed Central

    2011-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes prostaglandin E2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 μM, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis. PMID:21466167

  13. Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors.

    PubMed

    Morgunova, Ekaterina; Meining, Winfried; Illarionov, Boris; Haase, Ilka; Jin, Guangyi; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2005-03-01

    The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range. PMID:15723519

  14. Endogenous nitric oxide synthase inhibitors in the biology of disease: markers, mediators, and regulators?

    PubMed

    Caplin, Ben; Leiper, James

    2012-06-01

    The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring, and modulating circulating asymmetric dimethylarginine alone is inadequate. This review outlines the basic biochemistry and physiology of endogenous methylarginines, examines both the experimental and observational evidence for a role in disease pathogenesis, and examines the potential for therapeutic regulation of these molecules. PMID:22460557

  15. Endogenous nitric oxide synthase inhibitors in the biology of disease: markers, mediators and regulators?

    PubMed Central

    Caplin, Ben; Leiper, James

    2014-01-01

    The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino-acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring and/or modulating circulating asymmetric dimethylarginine alone is inadequate. This review will outline the basic biochemistry and physiology of endogenous methylarginines, examine both the experimental and observational evidence for a role in disease pathogenesis and examine the potential for therapeutic regulation of these molecules. PMID:22460557

  16. Acylation of naturally occurring and synthetic 1-deoxysphinganines by ceramide synthase. Formation of N-palmitoyl-aminopentol produces a toxic metabolite of hydrolyzed fumonisin, AP1, and a new category of ceramide synthase inhibitor.

    PubMed

    Humpf, H U; Schmelz, E M; Meredith, F I; Vesper, H; Vales, T R; Wang, E; Menaldino, D S; Liotta, D C; Merrill, A H

    1998-07-24

    Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro- and threo-2-amino, 3-hydroxy- (and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with Vmax/Km of 40-125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4-6 for the threo-isomers. Ceramide synthase also acylates AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a Vmax/Km of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro- and threo-isomers are acylated with the highest apparent Vmax/Km for the erythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins. PMID:9668088

  17. Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition.

    PubMed

    Moussian, Bernard; Letizia, Annalisa; Martnez-Corrales, Guillermo; Rotstein, Brbara; Casali, Andreu; Llimargas, Marta

    2015-01-01

    Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely expansion (exp) and rebuf (reb). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity. PMID:25617778

  18. Deciphering the Genetic Programme Triggering Timely and Spatially-Regulated Chitin Deposition

    PubMed Central

    Rotstein, Brbara; Casali, Andreu; Llimargas, Marta

    2015-01-01

    Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely expansion (exp) and rebuf (reb). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity. PMID:25617778

  19. Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

    PubMed

    Szabó, C; Bryk, R; Zingarelli, B; Southan, G J; Gahman, T C; Bhat, V; Salzman, A L; Wolff, D J

    1996-08-01

    1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2. GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 microM, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopterin. 3. Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD-1 cells were stimulated with appropriate mixtures of pro-inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11, 9, 1 and 30 microM, respectively). This suggests that uptake of GED may be cell type and species-dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4. GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 microM). Part of this relaxation was endothelium-dependent, but was not blocked by methylene blue (100 microM), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 microM and caused a slight inhibition of the relaxations at 100 microM. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5. In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium-dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6. Administration of GED (10 mg kg-1, i.p.) given at 2 h after LPS (120 mg kg-1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7. In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression. PMID:8842429

  20. Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

    PubMed Central

    Szab, C.; Bryk, R.; Zingarelli, B.; Southan, G. J.; Gahman, T. C.; Bhat, V.; Salzman, A. L.; Wolff, D. J.

    1996-01-01

    1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2. GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 microM, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopterin. 3. Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD-1 cells were stimulated with appropriate mixtures of pro-inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11, 9, 1 and 30 microM, respectively). This suggests that uptake of GED may be cell type and species-dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4. GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 microM). Part of this relaxation was endothelium-dependent, but was not blocked by methylene blue (100 microM), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 microM and caused a slight inhibition of the relaxations at 100 microM. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5. In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium-dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6. Administration of GED (10 mg kg-1, i.p.) given at 2 h after LPS (120 mg kg-1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7. In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression. Images Figure 8 Figure 9 Figure 10 PMID:8842429

  1. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  2. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    PubMed Central

    2013-01-01

    Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1100 muM) were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury. PMID:23363429

  3. Nitroarachidonic Acid, a Novel Peroxidase Inhibitor of Prostaglandin Endoperoxide H Synthases 1 and 2*

    PubMed Central

    Trostchansky, Andrs; Bonilla, Luca; Thomas, Christopher P.; ODonnell, Valerie B.; Marnett, Lawrence J.; Radi, Rafael; Rubbo, Homero

    2011-01-01

    Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H2. We have previously synthesized and chemically characterized nitroarachidonic acid (AANO2), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO2 with PGHS was analyzed. AANO2 inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO2 exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of kobs versus AANO2 concentrations showed a hyperbolic function with kinact = 0.045 s?1 and Ki*app = 0.019 ?m for PGHS-1 and kinact = 0.057 s?1 and Ki*app = 0.020 ?m for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO2 involves two sequential steps: an initial reversible binding event (described by Ki) followed by a practically irreversible event (Ki*app) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO2 were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO2 significantly decreased PGHS-1-dependent thromboxane B2 formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway. PMID:21266582

  4. Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Snoeberger, Ning-Shiuan Nicole

    Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

  5. The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

    SciTech Connect

    Korolev, S.; Ikeguchi, Y.; Skarina, T.; Beasley, S.; Arrowsmith, C.; Edwards, A.; Joachimiak, A.; Pegg, A. E.; Savchenko, A.; Pennsylvania State Univ. Coll. of Medicine; Milton S. Hershey Medical Center; Banting and Best Department of Medical Research; Univ. of Health Network

    2002-01-01

    Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Angstroms resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal {beta}-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

  6. Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery.

    PubMed

    Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pelà, Michela; Pinetti, Diego; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana

    2015-12-01

    Recently, octapeptide LSCQLYQR (LRp), reducing growth of cis-platinum (cDDP) resistant ovarian carcinoma cells by inhibiting the monomer-monomer interface of the human enzyme thymidylate synthase, has been identified. As the peptide is not able to cross the cell membrane it requires an appropriate delivery system. In this work the application of SLNs, biocompatible and efficient tools for the intracellular drug transport, applied especially for lipophilic drugs, was exploited for the delivery of the hydrophilic peptide LRp. SLNs formulated in the absence/presence of small amount of squalene showed dimensions below 150nm, negative zeta potential and good stability to the freeze-drying process. Even though the particles formulated with squalene exhibited a less ordered crystal lattice and a lower surface hydrophobicity, a rapid drug release from these nanocarriers occurred as a result of the relevant expulsion of the drug from the lipid core during lipid crystallization. On the contrary, SLNs formulated in the absence of squalene were able to incorporate more stably the peptide showing considerable cytotoxic effect on cDDP resistant C13* ovarian carcinoma cell line at concentration 50 times lower than that used previously with a marketed delivery system. From the cell cycle analysis by the propidium iodide test in SLNs-peptide treated cancer cells an increase of apoptosis percentage was observed, indicating that SLNs were able to carry efficiently the peptide until its enzymatic target. PMID:26433347

  7. Chitin Nanowhisker Aerogels

    PubMed Central

    Heath, Lindy; Zhu, Lifan; Thielemans, Wim

    2013-01-01

    Chitin nanowhiskers are structured into mesoporous aerogels by using the same benign process used previously in our group to make cellulose nanowhisker aerogels. The nanowhiskers are sonicated in water to form a hydrogel before solvent-exchange with ethanol and drying under supercritical CO2 (scCO2). Aerogels are prepared with various densities and porosities, relating directly to the initial chitin nanowhisker content. scCO2 drying enables the mesoporous network structure to be retained as well as allowing the gel to retain its initial dimensions. The chitin aerogels have low densities (0.0430.113 g cm?3), high porosities (up to 97 %), surface areas of up to 261 m2 g?1, and mechanical properties at the high end of other reported values (modulus between 7 and 9.3 MPa). The aerogels were further characterized by using X-ray diffraction, BET analysis, electron microscopy, FTIR, and thermogravimetric analysis. Characterization showed that the rod-like crystalline nature of the nanowhiskers was retained during the aerogel production process, making the aerogel truly an assembled structure of chitin nanocrystals. These aerogels also showed the lowest reported shrinkage during drying to date, with an average shrinkage of only 4 %. PMID:23335426

  8. A Novel Lumazine Synthase Inhibitor Derived from Oxidation of 1,3,6,8-Tetrahydroxy-2,7-naphthyridine to a Tetraazaperylenehexaone Derivative

    PubMed Central

    Zhang, Yanlei; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Georg, Gunda I.; Ye, Qi-Zhuang; Velde, David Vander; Fanwick, Phillip E.; Song, Yunlong; Cushman, Mark

    2008-01-01

    Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-Ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66 13 ?M in Tris buffer and 22 4 ?M in phosphate buffer. This is significantly more potent than the reactant (Ki 350 76 ?M, competitive inhibition), which had previously been identified as a lumazine synthase inhibitor by high-throughput screening. Ab initio calculations indicate that the meso form is slightly less stable than the enantiomeric form, and that the two forms interconvert rapidly at room temperature. PMID:17348709

  9. The impact of asymmetric dimethylarginine (ADAMA), the endogenous nitric oxide (NO) synthase inhibitor, to the pathogenesis of gastric mucosal damage.

    PubMed

    Szlachcic, Aleksandra; Krzysiek-Maczka, Gracjana; Pajdo, Robert; Targosz, Aneta; Magierowski, Marcin; Jasnos, Katarzyna; Drozdowicz, Danuta; Kwiecien, Slawomir; Brzozowski, Tomasz

    2013-01-01

    This review was designed to provide an update on the role of asymmetric arginine (ADMA), the endogenous inhibitor of nitric oxide (NO) synthase in the pathophysiology of the upper gastrointestinal (GI) tract. Numerous studies in the past confirmed that NO is a multifunctional endogenous gas molecule involved in most of the body organs' functional and metabolic processes including the regulation of gastrointestinal (GI) secretory functions, motility, maintenance of GI integrity, gastroprotection and ulcer healing. NO is metabolized from L-arginine by enzymatic reaction in the presence of constitutive NO synthase. In upper GI tract, NO acts as a potent vasodilator known to increase gastric mucosa blood flow, regulates the secretion of mucus and bicarbonate, inhibits the gastric secretion and protects the gastric mucosa against the damage induced by a variety of damaging agents and corrosive substances. In contrast, ADMA first time described by Vallance and coworkers in 1992, is synthesized by the hydrolysis of proteins containing methylated arginine amino acids located predominantly within the nucleus of cells. This molecule has been shown to competitively inhibit NO synthase suggesting its regulatory role in the functions of vascular endothelial cells and systemic circulation in humans and experimental animals. Nowadays, ADMA is a potentially important risk factor for coronary artery diseases and a marker of cardiovascular risk. Increased plasma levels of ADMA have been documented in several conditions that are characterized by endothelial dysfunction, including hypertension, hypercholesterolemia, hyperglycemia, renal failure and tobacco exposure. The role of ADMA in other systems including GI-tract has been so far less documented. Nevertheless, ADMA was shown to directly induce oxidative stress and cell apoptosis in gastric mucosal cells in vitro and to contribute to the inflammatory reaction associated with major human pathogen to gastric mucosa, Helicobacter pylori (H.pylori). Infection of gastric mucosa with this germ or H. pylori water extract led to marked increase in the plasma concentration of ADMA and significantly inhibited bicarbonate secretion, considered as one of the important components of upper GI-tract defense system. When administered to rodents, ADMA aggravated gastric mucosal lesions injury induced by cold stress, ethanol and indomethacin and this worsening effect on gastric lesions was accompanied by the significant increase in the plasma level of ADMA. This exaggeration of gastric lesions by ADMA was coincided with the inhibition of NO, the suppression of gastric blood flow and excessive release of proinflammatory cytokine TNF-α. This metabolic analog of L-arginine applied to rats was exposed to water immersion and restraint stress and ischemia-reperfusion, causing an elevation of plasma levels of ADMA and gastric MDA content, which is the marker of lipid peroxidation. These effects, including the rise in the plasma levels of ADMA in rats with stress and ischemia-reperfusion-induced gastric lesions, were attenuated by concomitant treatment with L-arginine, the substrate for NO-synthase, and superoxide dismutase (SOD), a reactive oxygen metabolite scavenger added to ADMA. We conclude that ADMA could be considered as an important factor contributing to the pathogenesis of gastric mucosal damage and inflammatory reaction in H. pylori-infected stomach due to inhibition of NO, suppression of GI microcirculation, and the proinflammatory and proapoptotic actions of this arginine analog. PMID:22950506

  10. Mechanisms of bacterial acetohydroxyacid synthase (AHAS) and specific inhibitors of Mycobacterium tuberculosis AHAS as potential drug candidates against tuberculosis.

    PubMed

    Gokhale, Kunal; Tilak, Bhargav

    2015-01-01

    On account of the ever increasing resistance of M.tuberculosis strains to orthodox therapy regimens, the task of combating tuberculosis becomes even more challenging. Therefore, there arises a need to isolate new drug targets and subsequently design specific inhibitors for the same. In bacteria, algae, plants and fungi, the synthesis of Branched Chain Amino Acids (BCAAs) is catalyzed by Acetohydroxyacid Synthases (AHAS) group of enzymes. Bacterial AHAS (EC 2.2.1.6) catalyzes the biosynthesis of isoleucine, leucine and valine by utilizing cofactors like Thiamin Diphosphate (ThDP), Flavin Adenine Dinucleotide (FAD) and a divalent metal cation (Usually Mg(2+)). The anabolic form of the enzyme which is presently under discussion consists of two subunits out of which one is catalytic while the other is regulatory in nature. The product of this enzyme catalyzed reaction is either 2-acetolactate or 2-aceto-2-hydroxybutyrate obtained from self-condensation of pyruvate or condensation of puruvate and 2-ketobutyrate, respectively. These are further converted to the BCAAs by a series of other enzymes. The step catalyzed by AHAS is the first in the entire cascade and hence can be selectively targeted for the inhibition of this pathway. M.tuberculosis AHAS, which is encoded by the ilvB and ilvN operons is structurally related to E.coli AHAS and has a similar function. Therefore, specific drugs belonging to the classes of sulfonylureas, imidazolinones and benzoyl esters can be used as inhibitors of M.tuberculosis AHAS which would consequently deplete the BCAA supply to the bacteria. Thus, efficient bacteriostasis can be achieved. PMID:25882218

  11. Synthesis and evaluation of new sulfur-containing L-arginine-derived inhibitors of nitric oxide synthase.

    PubMed

    Ichimori, K; Stuehr, D J; Atkinson, R N; King, S B

    1999-05-20

    A series of compounds (7, 8, 10-17, 23) containing new functional groups derived by the combination of the substrate, intermediate, product, and known inhibitors of nitric oxide synthase (NOS) were prepared and evaluated against NOS. While none of the compounds assayed acted as a nitric oxide-producing substrate, the sulfur-containing arginine derivatives 10-12 were competitive inhibitors of iNOS with Ki's of 202, 7, and 58 microM, respectively. Compound 11 demonstrated the greatest potency against NOS-mediated citrulline formation for each of the three isoforms with IC50's of 6. 7, 19.7, and 13 microM for nNOS, eNOS, and iNOS, respectively. Compounds 10-12 each demonstrated a slight selectivity for inhibition of the neuronal isoform compared to the endothelial and inducible isoforms. These compounds also influenced the NADPH oxidase activity and heme iron spin state in a manner similar to structurally related compounds. Compound 10, a thiocarbonyl-containing compound, decreased the NADPH oxidase activity of the enzyme (EC50 = 190 microM) and shifted the heme iron spin state toward a low-spin configuration, similar to that of L-thiocitrulline. Compounds 11 and 12, S-alkylthiocitrulline derivatives, decreased the NADPH oxidase activity of the enzyme (EC50 = 6.6 and 180 microM, respectively) and shifted the heme iron spin state toward a high-spin configuration, similar to that of L-S-methylisothiocitrulline. Carbonyl-containing amino acid (7, 8, 23) and non-amino acid (13-17) analogues did not interact well with the enzyme. PMID:10346937

  12. Nitric oxide synthase inhibitors that interact with both heme propionate and tetrahydrobiopterin show high isoform selectivity.

    PubMed

    Kang, Soosung; Tang, Wei; Li, Huiying; Chreifi, Georges; Martásek, Pavel; Roman, Linda J; Poulos, Thomas L; Silverman, Richard B

    2014-05-22

    Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3-8) were designed to probe the structure-activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors. PMID:24758147

  13. Nitric Oxide Synthase Inhibitors That Interact with Both Heme Propionate and Tetrahydrobiopterin Show High Isoform Selectivity

    PubMed Central

    2015-01-01

    Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3–8) were designed to probe the structure–activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors. PMID:24758147

  14. Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis

    PubMed Central

    Nardone, Glenn; Ikeda, Allison K.; Cunnington, Aubrey J.; Okebe, Joseph; Ebonyi, Augustine O.; Njie, Madi; Correa, Simon; Jayasooriya, Shamanthi; Casals-Pascual, Climent; Billker, Oliver; Conway, David J.; Walther, Michael; Ackerman, Hans

    2015-01-01

    Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis. PMID:26407009

  15. Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis.

    PubMed

    Chertow, Jessica H; Alkaitis, Matthew S; Nardone, Glenn; Ikeda, Allison K; Cunnington, Aubrey J; Okebe, Joseph; Ebonyi, Augustine O; Njie, Madi; Correa, Simon; Jayasooriya, Shamanthi; Casals-Pascual, Climent; Billker, Oliver; Conway, David J; Walther, Michael; Ackerman, Hans

    2015-09-01

    Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis. PMID:26407009

  16. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain.

    PubMed

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15?mg/kg(-1), i.p., 7 days); group treated with 7-NI (25?mg/kg(-1), i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels. PMID:26576217

  17. Expression of the protein inhibitor of nitric oxide synthase in the adult rat retina before and after optic nerve lesion.

    PubMed

    Dietz, Gunnar P H; Schott, Michael; Labes, Monika; Bähr, Mathias

    2005-05-20

    The molecular messenger nitric oxide (NO) not only serves a number of physiologic functions, but is also involved in the pathophysiology of neurodegeneration. It is produced by the nitric oxide synthase (NOS) isoenzymes. One of the many players regulating NOS activity is the Protein Inhibitor of NOS, PIN. To gain further insight into the mechanisms of NOS regulation and NO-mediated cell death after nerve trauma, we examined PIN expression in a standard model of lesion-induced neurodegeneration, the rat optic nerve transsection model. In both the axotomized retinae and the control retinae PIN expression was predominantly observed in the retinal ganglion cell layer. Optic nerve lesion did neither change the amount of PIN mRNA, as determined by in situ hybridization and real-time RT-PCR, nor did it change the amount of PIN as determined by immunohistochemistry and Western blot analysis. These results suggest that in our model, NOS activity is not regulated by altered PIN levels, which contributes to our understanding of apoptotic mechanisms in injured neurons. PMID:15893595

  18. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain

    PubMed Central

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15?mg/kg?1, i.p., 7 days); group treated with 7-NI (25?mg/kg?1, i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels. PMID:26576217

  19. Phytotoxicity of Acetohydroxyacid Synthase Inhibitors Is Not Due to Accumulation of 2-Ketobutyrate and/or 2-Aminobutyrate.

    PubMed Central

    Shaner, D. L.; Singh, B. K.

    1993-01-01

    Acetohydroxyacid synthase (AHAS) is the site of action of herbicides of different chemical classes, such as imidazolinones, sulfonylureas, and triazolopyrimidines. Inhibition of AHAS causes the accumulation of 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) (the transamination product of 2-KB), and it has been proposed that the phytotoxicity of these inhibitors is due to this accumulation. Experiments were done to determine the relationship between accumulation of 2-KB and 2-AB and the phytotoxicity of imazaquin to maize (Zea mays). Imazaquin concentrations that inhibit growth of maize plants also cause the accumulation of 2-KB and 2-AB in the shoots. Supplementation of imazaquin-treated plants with isoleucine reduced the pools of 2-KB and 2-AB in the plant but did not protect plants from the growth inhibitory effects of imazaquin. Conversely, feeding 2-AB to maize plants increased 2-KB and 2-AB pools to much higher levels than those observed in imazaquin-treated plants, yet such high pools of 2-KB and 2-AB in the plant had no significant effect on growth. These results conclusively demonstrate that growth inhibition following imazaquin treatment is not due to accumulation of 2-KB and/or 2-AB in plants. Changes in the amino acid profiles after treatment with imazaquin suggest that starvation for the branched-chain amino acids may be the primary cause of growth retardation of maize. PMID:12232015

  20. In vitro and ex vivo activity of new derivatives of acetohydroxyacid synthase inhibitors against Mycobacterium tuberculosis and non-tuberculous mycobacteria.

    PubMed

    Sohn, Hosung; Lee, Kil-Soo; Ko, Young-Kwan; Ryu, Jae-Wook; Woo, Jae-Choon; Koo, Dong-Wan; Shin, Sung-Jae; Ahn, Se-Jin; Shin, A-Rum; Song, Chang-Hwa; Jo, Eun-Kyeong; Park, Jeong-Kyu; Kim, Hwa-Jung

    2008-06-01

    Sulfometuron methyl (SM) is an inhibitor of acetohydroxyacid synthase (AHAS), the first common enzyme in the branched-chain amino acid biosynthetic pathway, and shows activity against Mycobacterium tuberculosis both in vitro and in vivo. To develop AHAS inhibitor derivatives with more potent activity, 100 sulfonylurea analogues were screened for antimycobacterial activity against M. tuberculosis and non-tuberculous mycobacteria (NTM), and then evaluated for intracellular activity using mouse macrophages. Three new compounds with antimycobacterial activity comparable with that of SM were identified. These compounds exhibit significant activity against intracellular M. tuberculosis (including the drug-resistant M. tuberculosis strains), and NTM Mycobacterium abscessus and Mycobacterium kansasii, respectively. PMID:18337064

  1. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics. PMID:26751161

  2. Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris.

    PubMed

    Ehrlich, Hermann; Kaluzhnaya, Oksana V; Brunner, Eike; Tsurkan, Mikhail V; Ereskovsky, Alexander; Ilan, Micha; Tabachnick, Konstantin R; Bazhenov, Vasilii V; Paasch, Silvia; Kammer, Martin; Born, Ren; Stelling, Allison; Galli, Roberta; Belikov, Sergei; Petrova, Olga V; Sivkov, Victor V; Vyalikh, Denis; Hunoldt, Sebastian; Wrheide, Gert

    2013-09-01

    This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to ?-chitin, known to be present in other animals, than to ?-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor. PMID:23831449

  3. Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform.

    PubMed Central

    Southan, G. J.; Zingarelli, B.; O'Connor, M.; Salzman, A. L.; Szab, C.

    1996-01-01

    1. The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2. AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 microM (EC50 values for NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine were 159 and > 1000 microM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may complete with L-arginine for its binding site on NOS. 3. The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4. Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5. These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6. In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than NG-methyl-L-arginine and NG-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7. AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8. High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9. In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10. In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS. PMID:8646406

  4. Selective Enhancement of the Uptake and Bioactivity of a TAT-conjugated Peptide Inhibitor of Glycogen Synthase Kinase-3

    PubMed Central

    Manceur, Aziza P; Driscoll, Brandon D; Sun, Wei; Audet, Julie

    2008-01-01

    The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2?Z,3?E)-6-bromoindirubin-3?-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types. PMID:19107119

  5. Plant availability and phytotoxicity of soil bound residues of herbicide ZJ0273, a novel acetolactate synthase potential inhibitor.

    PubMed

    Han, Ailiang; Yue, Ling; Li, Zheng; Wang, Haiyan; Wang, Yue; Ye, Qingfu; Lu, Long; Gan, Jay

    2009-11-01

    The plant availability and phytotoxicity of soil bound residues (BR) of herbicide ZJ0273, a novel acetolactate synthase (ALS) potential inhibitor, to rice (Oryza sativa L.) and corn (Zea mays L.) was investigated in three different soils including a Fluvio-marine yellow loamy soil (S(1)), a Red clayey soil (S(2)), and a Coastal saline soil (S(3)), using (14)C-labeling tracer and bioassay techniques. When soils were amended with BR at 0.6, 1.2 and 1.8 nmol g(-1), dose-dependent and significant inhibition was observed for rice seedlings within 14d after treatment, but no significant inhibition occurred to corn seedlings in the same treatment. Radioactive analysis of soil extracts following sequential extractions showed that the (14)C labeled residues of ZJ0273 were released from the amended soil BR upon planting. For example, when amended with 1.8 nmol g(-1), about 68.3%, 57.0%, and 61.1%, respectively, of the added BR were released in S(1), S(2), and S(3) planted with rice seedlings, whereas 38.9%, 32.7% and 32.6% became available for uptake in the corresponding soils planted with corn seedlings. The released compounds were identified as ZJ0273 and its degradation products M1 and M2, with M2 as the primary component. Bioassay on rice showed that concentration for 50% inhibition (IC(50)) of ZJ0273, M1, and M2 were 33.16, 1.93 and 0.49 microM, respectively. Therefore, BR formed after application of ZJ0273 may become available for plant uptake during rice cultivation and lead to phytotoxic effects, and the phytotoxicity is mainly caused by the release of the biologically active metabolite M2. This knowledge is valuable for designing crop rotation practices so that crop injury and yield losses due to carry-over herbicide phytotoxicity may be avoided. PMID:19732936

  6. CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

    PubMed

    Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-02-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  7. CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

    PubMed Central

    Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-01-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  8. The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

    PubMed

    Gocmez, Semil Selcen; Yazir, Yusufhan; Sahin, Deniz; Karadenizli, Sabriye; Utkan, Tijen

    2015-04-01

    Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats. PMID:25636602

  9. Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors.

    PubMed

    Li, Ya-Li; Qi, Xiang-Yu; Jiang, Hui; Deng, Xiao-Dong; Dong, Yan-Ping; Ding, Ting-Bo; Zhou, Lu; Men, Peng; Chu, Yong; Wang, Ren-Xiao; Jiang, Xian-Cheng; Ye, De-Yong

    2015-09-15

    Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 ?M in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 ?M in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors. PMID:26314925

  10. Use of bacterial surrogates as a tool to explore antimalarial drug interaction: Synergism between inhibitors of malarial dihydrofolate reductase and dihydropteroate synthase.

    PubMed

    Talawanich, Yuwadee; Kamchonwongpaisan, Sumalee; Sirawaraporn, Worachart; Yuthavong, Yongyuth

    2015-09-01

    Interaction between antimalarial drugs is important in determining the outcome of chemotherapy using drug combinations. Inhibitors of dihydrofolate reductase (DHFR) such as pyrimethamine and of dihydropteroate synthase (DHPS) such as sulfa drugs are known to have synergistic interactions. However, studies of the synergism are complicated by the fact that the malaria parasite can also salvage exogenous folates, and the salvage may also be affected by the drugs. It is desirable to have a convenient system to study interaction of DHFR and DHPS inhibitors without such complications. Here, we describe the use of Escherichia coli transformed with malarial DHFR and DHPS, while its own corresponding genes have been inactivated by optimal concentration of trimethoprim and genetic knockout, respectively, to study the interaction of the inhibitors. Marked synergistic effects are observed for all combinations of pyrimethamine and sulfa inhibitors in the presence of trimethoprim. At 0.05μM trimethoprim, sum of fractional inhibitory concentrations, ΣFIC of pyrimethamine with sulfadoxine, pyrimethamine with sulfathiazole, pyrimethamine with sulfamethoxazole, and pyrimethamine with dapsone are in the range of 0.24-0.41. These results show synergism between inhibitors of the two enzymes even in the absence of folate transport and uptake. This bacterial surrogate system should be useful as a tool for assessing the interactions of drug combinations between the DHFR and DHPS inhibitors. PMID:25997881

  11. Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.

    PubMed

    Cabrera-Salazar, Mario A; Deriso, Matthew; Bercury, Scott D; Li, Lingyun; Lydon, John T; Weber, William; Pande, Nilesh; Cromwell, Mandy A; Copeland, Diane; Leonard, John; Cheng, Seng H; Scheule, Ronald K

    2012-01-01

    Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases. PMID:22912851

  12. An NMR Biochemical Assay for Fragment-Based Drug Discovery: Evaluation of an Inhibitor Activity on Spermidine Synthase of Trypanosoma cruzi.

    PubMed

    Yamasaki, Kazuhiko; Tani, Osamu; Tateishi, Yukihiro; Tanabe, Eiki; Namatame, Ichiji; Niimi, Tatsuya; Furukawa, Koji; Sakashita, Hitoshi

    2016-03-10

    Although NMR in fragment-based drug discovery is utilized almost exclusively to evaluate physical binding between molecules, it should be also a powerful tool for biochemical assay, evaluating inhibitory effect of compounds on enzymatic activity. Time-dependent spectral change in real-time monitoring or inhibitor concentration-dependent spectral change after constant-time reaction was processed by factor analysis, by which reaction rate or IC50 value was obtained. Applications to spermidine synthase of Trypanosoma cruzi, which causes Chagas disease, are described. PMID:26881725

  13. Chitin Deacetylases: Properties and Applications

    PubMed Central

    Zhao, Yong; Park, Ro-Dong; Muzzarelli, Riccardo A. A.

    2010-01-01

    Chitin deacetylases, occurring in marine bacteria, several fungi and a few insects, catalyze the deacetylation of chitin, a structural biopolymer found in countless forms of marine life, fungal cell and spore walls as well as insect cuticle and peritrophic matrices. The deacetylases recognize a sequence of four GlcNAc units in the substrate, one of which undergoes deacetylation: the resulting chitosan has a more regular deacetylation pattern than a chitosan treated with hot NaOH. Nevertheless plain chitin is a poor substrate, but glycolated, reprecipitated or depolymerized chitins are good ones. The marine Vibrio sp. colonize the chitin particles and decompose the chitin thanks to the concerted action of chitinases and deacetylases, otherwise they could not tolerate chitosan, a recognized antibacterial biopolymer. In fact, chitosan is used to prevent infections in fishes and crustaceans. Considering that chitin deacetylases play very important roles in the biological attack and defense systems, they may find applications for the biological control of fungal plant pathogens or insect pests in agriculture and for the biocontrol of opportunistic fungal human pathogens. PMID:20161969

  14. Ceestatin, a Novel Small Molecule Inhibitor of Hepatitis C Virus Replication, Inhibits 3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase

    PubMed Central

    Peng, Lee F.; Schaefer, Esperance A. K.; Maloof, Nicole; Skaff, Andrew; Berical, Andrew; Belon, Craig A.; Heck, Julie A.; Lin, Wenyu; Frick, David N.; Allen, Todd M.; Miziorko, Henry M.; Schreiber, Stuart L.

    2011-01-01

    Background.?Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library. Methods.?To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. Results.?Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatins anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. Conclusions.?Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act. PMID:21791663

  15. Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin A, a nitrated phytotoxin produced by Streptomyces spp.

    PubMed

    Wach, Michael J; Kers, Johan A; Krasnoff, Stuart B; Loria, Rosemary; Gibson, Donna M

    2005-02-01

    Evidence for the involvement of a bacterial nitric oxide synthase (NOS) in the biosynthesis of a phytotoxin is presented. Several species of Streptomyces bacteria produce secondary metabolites with unusual nitrogen groups, such as thaxtomin A (ThxA), which contains a nitroindole moiety. ThxA is a phytotoxin made by three pathogenic Streptomyces species that cause common scab of potato. All three species possess a gene homologous to the oxygenase domain of murine inducible NOS, and this gene, nos, is essential for normal levels of ThxA production. We grew Streptomyces turgidiscabies in the presence of several known NOS inhibitors and a nitric oxide (NO) scavenger to determine their effect on ThxA production. The NO scavenger (CPTIO) and four NOS inhibitors (NAME, NMMA, AG, and 7-NI) reduced ThxA production without affecting bacterial growth. A strain of S. turgidiscabies from which the nos gene had been deleted was grown in the presence of three NO donors (DEANO, SIN, and SNAP), and all three partially restored ThxA production. Our data suggest that bacterial nitric oxide synthases may, at least in part, produce NO for biosynthetic purposes, rather than for cellular signaling, as they do in mammals. PMID:15631947

  16. A novel lumazine synthase inhibitor derived from oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine to a tetraazaperylenehexaone derivative.

    PubMed

    Zhang, Yanlei; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Georg, Gunda I; Ye, Qi-Zhuang; Vander Velde, David; Fanwick, Phillip E; Song, Yunlong; Cushman, Mark

    2007-04-13

    Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66+/-13 microM in Tris buffer and 22+/-4 microM in phosphate buffer. This is significantly more potent than the reactant (Ki 350+/-76 microM, competitive inhibition), which had previously been identified as a lumazine synthase inhibitor by high-throughput screening. Ab initio calculations indicate that the meso form is slightly less stable than the enantiomeric form, and that the two forms interconvert rapidly at room temperature. PMID:17348709

  17. iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats

    PubMed Central

    Kaneki, Masao; Fukushima, Yuji; Shinozaki, Shohei; Fukaya, Makiko; Habiro, Mayu; Shimizu, Nobuyuki; Chang, Kyungho; Yasuhara, Shingo; Martyn, J.A. Jeevendra

    2014-01-01

    Objectives Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3β activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3β activity in skeletal muscle of burned rats. Materials/Methods Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160–190 g) by immersing the back of the trunk for 15 sec and the abdomen for 8 sec in 80°C water. Burned and sham-burned rats were treated with L-NIL (60 mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3β activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3β and its endogenous substrate, glycogen synthase. Results GSK-3β activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3β activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3β activity in sham-burned rats. Conclusions Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3β activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3β. PMID:22995863

  18. Novel Inhibitors of Ornithine Decarboxylase of Leishmania Parasite (LdODC): The Parasite Resists LdODC Inhibition by Overexpression of Spermidine Synthase.

    PubMed

    Das, Mousumi; Singh, Shalini; Dubey, Vikash Kumar

    2016-03-01

    Ornithine decarboxylase (LdODC), a key enzyme in polyamine biosynthesis in Leishmania donovani, catalyzes the conversion of ornithine to putrescine that is finally used for synthesis of spermidine and other polyamines. Inhibition of ornithine decarboxylase is likely to deplete the parasite trypanothione and may result in increased reactive oxygen species (ROS). Sequence as well as structure of LdODC and human ODC shows significant difference; therefore, we have identified novel specific inhibitors of LdODC. These inhibitors are able to inhibit recombinant LdODC and decrease intracellular putrescine concentration showing target specificity. The Ki values of LdODC inhibition do not correlate with IC50 values in Leishmania promastigote possibly due to different stability/pharmacokinetics. These inhibitors, except compound M-5, have only minor effect on Leishmania promastigotes, and IC50 values are several folds higher as compared to Ki values. In case of compound M-5, IC50 value is less than Ki value indicating that the compound may have additional targets. Our studies suggest that the parasite resists these LdODC inhibitors by overexpression of spermidine synthase mRNA. PMID:26362015

  19. An Innovative Strategy for Dual Inhibitor Design and Its Application in Dual Inhibition of Human Thymidylate Synthase and Dihydrofolate Reductase Enzymes

    PubMed Central

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang ping; Lee, Keun Woo

    2013-01-01

    Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS) and human dihydrofolate reductase (hDHFR). These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs. PMID:23577115

  20. Novel aldosterone synthase inhibitors with extended carbocyclic skeleton by a combined ligand-based and structure-based drug design approach.

    PubMed

    Lucas, Simon; Heim, Ralf; Negri, Matthias; Antes, Iris; Ries, Christina; Schewe, Katarzyna E; Bisi, Alessandra; Gobbi, Silvia; Hartmann, Rolf W

    2008-10-01

    Pharmacophore modeling of a series of aldosterone synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)-N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors (e.g., 17, IC50 = 2.7 nM) with pronounced selectivity toward the most important steroidogenic and hepatic CYP enzymes. PMID:18763754

  1. Glycogen synthase kinase 3 inhibitors induce the canonical WNT/?-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

    PubMed Central

    Chen, Eleanor Y.; DeRan, Michael T.; Ignatius, Myron S.; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M.; Lobbardi, Riadh M.; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M.

    2014-01-01

    Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/?-catenin pathway, recombinant WNT3A and stabilized ?-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/?-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/?-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/?-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

  2. Glycogen synthase kinase 3 inhibitors induce the canonical WNT/?-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma.

    PubMed

    Chen, Eleanor Y; DeRan, Michael T; Ignatius, Myron S; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M; Lobbardi, Riadh M; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M

    2014-04-01

    Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/?-catenin pathway, recombinant WNT3A and stabilized ?-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/?-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/?-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/?-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

  3. In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

    PubMed Central

    Urbina, Julio A.; Concepcion, Juan Luis; Caldera, Aura; Payares, Gilberto; Sanoja, Cristina; Otomo, Takeshi; Hiyoshi, Hironobu

    2004-01-01

    Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with Ki values in the low nanomolar to subnanomolar range in the absence or presence of 20 ?M inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease. PMID:15215084

  4. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    PubMed

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β. PMID:26610618

  5. Design and synthesis of N-2,6-difluorophenyl-5-methoxyl-1,2,4-triazolo[1,5-a]-pyrimidine-2-sulfonamide as acetohydroxyacid synthase inhibitor.

    PubMed

    Chen, Chao-Nan; Lv, Li-Li; Ji, Feng-Qin; Chen, Qiong; Xu, Hui; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

    2009-04-15

    Triazolopyrimidine-2-sulfonamide belongs to a herbicide group called acetohydroxyacid synthase inhibitors. With the aim to discover new triazolopyrimidine sulfonanilide compounds with high herbicidal activity and faster degradation rate in soil, the methyl group of Flumetsulam (FS) was modified into a methoxy group to produce a new herbicidal compound, N-2,6-difluorophenyl-5-methoxy-1,2,4-triazolo[1,5-a]pyrimidine-2-sulfonamide (experimental code: Y6610). The enzymatic kinetic results indicated that compound Y6610 and FS have k(i) values of 3.31x10(-6) M and 3.60x10(-7) M against Arabidopsis thaliana AHAS, respectively. The 10-fold lower enzyme-inhibiting activity of Y6610 was explained rationally by further computational simulations and binding free energy calculations. In addition, compound Y6610 was found to display the same level in vivo post-emergent herbicidal activity as FS against some broad-leaf weeds and good safety to rice, maize, and wheat at the dosages of 75-300 gai/ha. Further determination of the half-lives in soil revealed that the half-life in soil of Y6610 is 3.9 days shorter than that of FS. The experimental results herein showed that compound Y6610 could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study. PMID:19342247

  6. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    SciTech Connect

    Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  7. Discovery of depsides and depsidones from lichen as potent inhibitors of microsomal prostaglandin E2 synthase-1 using pharmacophore models.

    PubMed

    Bauer, Julia; Waltenberger, Birgit; Noha, Stefan M; Schuster, Daniela; Rollinger, Judith M; Boustie, Joel; Chollet, Marylene; Stuppner, Hermann; Werz, Oliver

    2012-12-01

    Nature in silico: Virtual screening using validated pharmacophore models identified lichen depsides and depsidones as potential inhibitors of mPGES-1, an emerging target for NSAIDs. Evaluation of the virtual hits in a cell-free assay revealed physodic acid and perlatolic acid as potent inhibitors of mPGES-1 (IC(50) = 0.4 and 0.43 ?M, respectively), indicating that these natural products have potential as novel anti-inflammatory agents. PMID:23109349

  8. First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin.

    PubMed

    Kaya, Murat; Baublys, Vykintas; Šatkauskienė, Ingrida; Akyuz, Bahar; Bulut, Esra; Tubelytė, Vaida

    2015-08-01

    Chitin immediately suggests the representatives of the kingdom Fungi, as well as such phyla as Annelida, Mollusca, Porifera, Cnidaria and, mostly, Arthropoda. Although Bryozoa also represents a chitin-containing phylum, no study has been developed yet on the isolation or characterization of the chitin from it. In this study, physiochemical properties of the chitin isolated from Plumatella repens belonging to the phylum Bryozoa was determined for the first time. The chitin structure was also studied comparatively by isolating chitin from an insect species (Palomena prasina) of the phylum Arthropoda, and Fomes fomentarius belonging to the kingdom Fungi. It was observed that the bryozoan chitin was in the α form, as in the arthropod and fungal chitins. The chitin contents in the dry weight of the bryozoan, fungal and insect species were observed to be 13.3%, 2.4%, and 10.8%, respectively. The insect chitin exhibited the highest thermal stability followed by that of the bryozoan and then the fungal chitins. Surface morphologies reveal that the insect and bryozoan chitins were composed of nano fibre and pore structures, whereas the fungal chitin had no pores or fibres. The crystallinity of the insect chitin (CrI=84.9%) was higher than the bryozoan (CrI=60.1%) and fungal chitins (CrI=58.5%). PMID:25940531

  9. Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Stanton, Benjamin Z.; Igarashi, Jotaro; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early “ADME/Tox” considerations and provides a basic platform for medicinal chemistry-driven efforts. PMID:18321097

  10. Structure-Based Design and Synthesis of Nω-Nitro-L-Arginine-Containing Peptidomimetics as Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural Water

    PubMed Central

    Seo, Jiwon; Igarashi, Jotato; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2008-01-01

    The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N′-nitroguanidine (1) and 4-N-(Nω-nitro-L-argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). Based on this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3–5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 4 and 5). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures. PMID:17425297

  11. The effect of an specific inducible NO synthase inhibitor, S-methylisothiourea hemisulfate on cisplatin-induced nephrotoxicity; gender-related differences

    PubMed Central

    Ghayyoomi, Mansooreh; Soltani, Nepton; Nematbakhsh, Mehdi; Moslemi, Fatemeh; Talebi, Ardeshir; Shirdavani, Soheila; Razmjoo, Farzaneh

    2015-01-01

    Backgrounds: It has been previously demonstrated that the increase of nitric oxide (NO) level may promote cisplatin (CP)-induced nephrotoxicity. The aim of this study was to investigate the role of inducible NO synthase (iNOS) inhibitor to prevent CP-induced nephrotoxicity. Materials and Methods: Four groups of male and four groups of female rats were treated daily with vehicle, S-methylisothiourea hemisulfate (SMT) as a selective iNOS inhibitor (5 mg/kg/twice a day), CP (2.5 mg/kg/day), and CP + SMT for 6 days. Then, all animals were sacrificed and the serum levels of creatinine (Cr), blood urea nitrogen (BUN), nitrite, and malondialdehyde (MDA) were measured. The kidney was removed immediately for histopathological study. Results: Our results showed that inhibition of iNOS by SMT could make different response in male and female animals. SMT therapy in male animals decreased serum BUN, Cr, nitrite, and MDA levels; and it also protected kidney against CP-induced nephrotoxicity. Conclusion: It is concluded that decrease in NO production by SMT has a beneficial effect on reducing CP-induced nephrotoxicity in male. However, such beneficial effect was not observed in female animals. PMID:26322278

  12. Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone, selective inhibitors of 3-ketoacyl-CoA synthases.

    PubMed

    Tresch, Stefan; Heilmann, Monika; Christiansen, Nicole; Looser, Ralf; Grossmann, Klaus

    2012-04-01

    The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC(50) values in ?M range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone. PMID:22284369

  13. The marine natural-derived inhibitors of glycogen synthase kinase-3β phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling

    PubMed Central

    Khanfar, Mohammad A.; Asal, Bilal Abu; Mudit, Mudit; Kaddoumi, Amal; El Sayed, Khalid A.

    2009-01-01

    The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2) showed potent in vitro and in vivo anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT, spheroid disaggregation, and in mice models. To explore a possible molecular target of PMHs, the most potent synthetic analogue 2 has been virtually screened against various protein kinases. Molecular modeling study has shown that 2 can be successfully docked within the binding pocket of glycogen synthase kinase-3beta (GSK-3β) similar to the well-known GSK-3β inhibitor I-5. Several PMHs showed potent in vitro GSK-3β inhibitory activity with an IC50 range of 4–20 µM. The most potent analogue 3 showed a significant increase in liver glycogen level at the 5, 15, and 25 mg/kg dose levels, in vivo. Pharmacophore model was built and validated using in-house database of active and inactive GSK-3β inhibitors. The GSK-3β inhibitory activity of PMHs entitles them to be potential leads for the treatment of cancer, Alzheimer’s disease, bipolar disorders, stroke, different tau pathologies, and type-2 diabetes. PMID:19616957

  14. In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

    PubMed

    Lucas, Simon; Heim, Ralf; Ries, Christina; Schewe, Katarzyna E; Birk, Barbara; Hartmann, Rolf W

    2008-12-25

    Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats. PMID:19049427

  15. Overcoming undesirable CYP1A2 inhibition of pyridylnaphthalene-type aldosterone synthase inhibitors: influence of heteroaryl derivatization on potency and selectivity.

    PubMed

    Heim, Ralf; Lucas, Simon; Grombein, Cornelia M; Ries, Christina; Schewe, Katarzyna E; Negri, Matthias; Mller-Vieira, Ursula; Birk, Barbara; Hartmann, Rolf W

    2008-08-28

    Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats. PMID:18672861

  16. The marine natural-derived inhibitors of glycogen synthase kinase-3beta phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling.

    PubMed

    Khanfar, Mohammad A; Asal, Bilal Abu; Mudit, Mudit; Kaddoumi, Amal; El Sayed, Khalid A

    2009-08-15

    The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2) showed potent in vitro and in vivo anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT, spheroid disaggregation, and in mice models. To explore a possible molecular target of PMHs, the most potent synthetic analogue 2 has been virtually screened against various protein kinases. Molecular modeling study has shown that 2 can be successfully docked within the binding pocket of glycogen synthase kinase-3beta (GSK-3beta) similar to the well-known GSK-3beta inhibitor I-5. Several PMHs showed potent in vitro GSK-3beta inhibitory activity with an IC(50) range of 4-20microM. The most potent analogue 3 showed a significant increase in liver glycogen level at the 5, 15, and 25mg/kg dose levels, in vivo. Pharmacophore model was built and validated using in-house database of active and inactive GSK-3beta inhibitors. The GSK-3beta inhibitory activity of PMHs entitles them to be potential leads for the treatment of cancer, Alzheimer's disease, bipolar disorders, stroke, different tau pathologies, and type-2 diabetes. PMID:19616957

  17. Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity

    SciTech Connect

    Zhao, Ying; Hammoudeh, Dalia; Yun, Mi-Kyung; Qi, Jianjun; White, Stephen W.; Lee, Richard E.

    2012-05-29

    Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

  18. Chitin deposition on the embryonic cuticle of Rhodnius prolixus: the reduction of CHS transcripts by CHS-dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph.

    PubMed

    Souza-Ferreira, Paula S; Mansur, Juliana F; Berni, Matheus; Moreira, Monica F; dos Santos, Roberto Eizemberg; Arajo, Helena M Marcolla; de Souza, Wanderley; Ramos, Isabela B; Masuda, Hatisaburo

    2014-08-01

    In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R.prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R.prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R.prolixus females lasted up to 3 egg-laying cycles (?100 days), pointing to R.prolixus as auseful model for developmental studies. PMID:24412274

  19. Docking studies of flavonoid compounds as inhibitors of ?-ketoacyl acyl carrier protein synthase I (Kas I) of Escherichia coli.

    PubMed

    Sabbagh, Ghalia; Berakdar, Noura

    2015-09-01

    Escherichia coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), traveler's diarrhea and other clinical infections such as neonatal meningitis and pneumonia. The fatty acid biosynthesis is essential for the bacterial viability and growth. There are three types of ?-ketoacyl acyl carrier protein synthase (KAS) which are important for overcoming the bacterial resistance problem. ?-ketoacyl acyl carrier protein synthase I (KAS I) is member of the condensing enzyme family, which is a key catalyst in bacterial fatty acid biosynthesis, and thus an attractive target for novel antibioticsis related to the elongation of unsaturated fatty acids in bacterial fatty acid synthesis and can be a good therapeutic target of designing novel antibiotics. In this report, we performed docking study of E. coli (KAS I) and 50 flavonoids. Out of these 50 flavonoids, there are two compounds, genistein and isorhamnetin, that showed the superior binding energy while fully satisfying the conditions of drug likeliness. The predicted binding energy of genistein and isorhamnetin toward KAS I are -135.76kcal/mol and -132.42kcal/mol, respectively. These energies favorably compare to the biding energy of known drugs thiolactomicin and cerulenin that are -90.26kcal/mol and -99.64kcal/mol, respectively. The method used was docking with the selected E. coli (KAS I-PDB ID-1FJ4) using iGemdock. This was also found to obey the Lipinski's guidelines of five and to show the drug likeliness and bioavailability. PMID:26292066

  20. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    SciTech Connect

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  1. Structure Determination of Glycogen Synthase Kinase-3 from Leishmania major and Comparative Inhibitor Structure-Activity Relationships with Trypanosoma brucei GSK-3

    PubMed Central

    Ojo, Kayode K.; Arakaki, Tracy L.; Napuli, Alberto J.; Inampudi, Krishna K.; Keyloun, Katelyn R.; Zhang, Li; Hol, Wim G.J.; Verlinde, Christophe L.M.J.; Merritt, Ethan A.; Van Voorhis, Wesley C.

    2011-01-01

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

  2. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3.

    PubMed

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G J; Verlinde, Christophe L M J; Merritt, Ethan A; Van Voorhis, Wesley C

    2011-04-01

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 ? resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

  3. Identification of novel membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitors with anti-influenza activities in vitro.

    PubMed

    Park, Ji Hoon; Park, Eun Beul; Lee, Jae Yeol; Min, Ji-Young

    2016-01-22

    Influenza A virus (IAV) is a major public health concern that leads to high morbidity and mortality worldwide. Despite various vaccination programs and development of drugs targeting essential viral proteins, the emergence of drug-resistant variants has been frequently reported and the therapeutic options are limited. Because exaggerated inflammation is considered as an important factor in disease pathogenesis, immunomodulatory agents that effectively suppress cytokine responses are needed for the treatment of IAV infection. Membrane-associated prostaglandin E synthase-1 (mPGES-1) is an enzyme responsible for the production of prostaglandin E2 (PGE2) that is the best-characterized immune modulatory lipid in vitro and in vivo models of inflammation. In the present study, we tested the anti-influenza activities of mPGES-1 inhibitors, using a phenotype-based assay involving image analyses. Seven primary hits among 49 compounds targeting mPGES-1 exhibited anti-influenza activities against A/Puerto Rico/8/1934 (H1N1) in a dose-dependent manner. The most effective hit, MPO-0047, suppressed influenza-induced p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) activation. We also showed that mRNA levels of TNF-α, IL-8, CCL5/RANTES, and CXCL10/IP-10 were significantly reduced by the treatment of influenza-infected cells with MPO-0047. Exogenous PGE2 reversed the inhibitory effects of MPO-0047. Our results showed that this selective mPGES-1 inhibitor has anti-influenza effects by inhibiting PGE2 production, which suppresses the induction of pro-inflammatory genes. Taken together our data revealed that mPGES-1 inhibitor has the potential for further development as an influenza therapeutic agent. PMID:26673392

  4. Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3? inhibitor, SB216763

    PubMed Central

    Brooks, Morgan M.; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy

    2013-01-01

    Purpose Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3? (GSK-3?) in regulating mMPT. Using direct inhibition of GSK-3? with the GSK-3? inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Methods Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O2) followed by exposure to atmospheric oxygen (approximately 21% O2). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Results Treatment of HLE-B3 cells with SB216763 (12 M), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Conclusions Inhibition of GSK-3? activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3? activity by the GSK-3? inhibitor SB216763 provides positive protection against mitochondrial depolarization. PMID:23825920

  5. Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

    PubMed

    Cioccoloni, Giorgia; Bonmassar, Laura; Pagani, Elena; Caporali, Simona; Fuggetta, Maria Pia; Bonmassar, Enzo; D'Atri, Stefania; Aquino, Angelo

    2015-08-01

    Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 M orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis. PMID:26035182

  6. Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice.

    PubMed

    Nietupski, Jennifer B; Pacheco, Joshua J; Chuang, Wei-Lien; Maratea, Kimberly; Li, Lingyun; Foley, Joseph; Ashe, Karen M; Cooper, Christopher G F; Aerts, Johannes M F G; Copeland, Diane P; Scheule, Ronald K; Cheng, Seng H; Marshall, John

    2012-04-01

    Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction. PMID:22366055

  7. Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon1[W

    PubMed Central

    Iwakami, Satoshi; Endo, Masaki; Saika, Hiroaki; Okuno, Junichi; Nakamura, Naoki; Yokoyama, Masao; Watanabe, Hiroaki; Toki, Seiichi; Uchino, Akira; Inamura, Tatsuya

    2014-01-01

    Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon. PMID:24760819

  8. 16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana.

    PubMed

    Roy, Arnab; Roberts, Frank G; Wilderman, P Ross; Zhou, Ke; Peters, Reuben J; Coates, Robert M

    2007-10-17

    The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(II)- and Pb(IV)-induced cyclizations onto the Delta12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC50 = 1 x 10-7 and 1 x 10-6 M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl diphosphate substrate (Km = 3 x 10-7 M). Large enhancements of binding affinities (IC50 = 4 x 10-9 and 2 x 10-8 M) were observed in the presence of 1 mM pyrophosphate, which is consistent with a tightly bound ent-beyeranyl+/pyrophosphate- ion pair intermediate in the cyclization-rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 x 10-5 M) exhibited by ent-beyeran-16-exo-yl diphosphate (11) and its failure to undergo bridge rearrangement to kaurene appear to rule out the covalent diphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-16-yl carbocation with potential applications as an active site probe for the various ent-diterpene cyclases and as a novel, selective inhibitor of gibberellin biosynthesis in plants. PMID:17892288

  9. 16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: Potent Mechanism-based Inhibitors of Recombinant ent-Kaurene Synthase from Arabidopsis thaliana1

    PubMed Central

    Roy, Arnab; Roberts, Frank G.; Wilderman, P. Ross; Zhou, Ke; Peters, Reuben J.; Coates, Robert M.

    2013-01-01

    The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to > 1,000 known diterpenes. (Scheme 1) To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues, 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13), by means of Hg(II)- and Pb(IV)-induced cyclizations onto the ?12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco 16-nor carbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC = 1 10 ?7 and 1 10?6 50 M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl diphosphate substrate (Km = 3 10?7 M). Large enhancements of binding affinities (IC50 = 4 10?9 and 2 10?8 M) were observed in the presence of 1 mM pyrophosphate which is consistent with a tightly bound ent-beyeranyl+/pyrophosphate? ion pair intermediate in the cyclization-rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 10?5 M) exhibited by ent-beyeran-16 exo-yl diphosphate (11), and its failure to undergo bridge rearrangement to kaurene, appear to rule out the covalent diphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-16-yl carbocation with potential applications as an active site probe for the various ent- diterpene cyclases, and as a novel, selective inhibitor of gibberellin biosynthesis in plants. PMID:17892288

  10. Alkaloids as Inhibitors of Malate Synthase from Paracoccidioides spp.: Receptor-Ligand Interaction-Based Virtual Screening and Molecular Docking Studies, Antifungal Activity, and the Adhesion Process

    PubMed Central

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Ceclia Maria Alves; Kato, Luclia; Freitas, Carla dos Santos; Giannini, Maria Jos Soares Mendes; da Silva, Julhiany de Ftima; Soares, Clia Maria de Almeida

    2015-01-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify ?-carboline moiety as a standard key structure. The compounds with ?-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of ?-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four ?-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. PMID:26124176

  11. Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon.

    PubMed

    Iwakami, Satoshi; Endo, Masaki; Saika, Hiroaki; Okuno, Junichi; Nakamura, Naoki; Yokoyama, Masao; Watanabe, Hiroaki; Toki, Seiichi; Uchino, Akira; Inamura, Tatsuya

    2014-04-23

    Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon. PMID:24760819

  12. Chitin-Like Molecules Associate with Cryptococcus neoformans Glucuronoxylomannan To Form a Glycan Complex with Previously Unknown Properties

    PubMed Central

    Ramos, Caroline L.; Fonseca, Fernanda L.; Rodrigues, Jessica; Guimares, Allan J.; Cinelli, Leonardo P.; Miranda, Kildare; Nimrichter, Leonardo; Casadevall, Arturo; Travassos, Luiz R.

    2012-01-01

    In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-?). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties. PMID:22562469

  13. A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

    PubMed

    Liu, Weitang; Yuan, Guohui; Du, Long; Guo, Wenlei; Li, Lingxu; Bi, Yaling; Wang, Jinxin

    2015-01-01

    Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation. PMID:25619909

  14. Chitin nanofibers: preparations, modifications, and applications

    NASA Astrophysics Data System (ADS)

    Ifuku, Shinsuke; Saimoto, Hiroyuki

    2012-05-01

    Chitin nanofibers are prepared from the exoskeletons of crabs and prawns by a simple mechanical treatment after the removal of proteins and minerals. The obtained nanofibers have fine nanofiber networks with a uniform width of approximately 10-20 nm and a high aspect ratio. The method used for chitin-nanofiber isolation is also successfully applied to the cell walls of mushrooms. They form a complex with glucans on the fiber surface. A grinder, a Star Burst atomization system, and a high speed blender are all used in the mechanical treatment to convert chitin to nanofibers. Mechanical treatment under acidic conditions is the key to facilitate fibrillation. At pH 3-4, the cationization of amino groups on the fiber surface assists nano-fibrillation by electrostatic repulsive force. By applying this finding, we also prepared chitin nanofibers from dry chitin powder. Chitin nanofibers are acetylated to modify their surfaces. The acetyl DS can be controlled from 1 to 3 by changing the reaction time. An acetyl group is introduced heterogeneously from the surface to the core. Nanofiber morphology is maintained even in the case of high acetyl DS. Optically transparent chitin nanofiber composites are prepared with 11 different types of acrylic resins. Due to the nano-sized structure, all of the composites are highly transparent. Chitin nanofibers significantly increase the Young's moduli and the tensile strengths and decrease the thermal expansion of all acrylic resins due to the reinforcement effect of chitin nanofibers. Chitin nanofibers show chiral separation ability. The chitin nanofiber membrane transports the d-isomer of glutamic acid, phenylalanine, and lysine from the corresponding racemic amino acid mixtures faster than the corresponding l-isomer. The chitin nanofibers improve clinical symptoms and suppress ulcerative colitis in a DSS-induced mouse model of acute ulcerative colitis. Moreover, chitin nanofibers suppress myeloperoxidase activation in the colon and decrease serum interleukin-6 concentrations.

  15. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

  16. Activity of chitin deacetylase from Colletotrichum gloeosporioides on chitinous substrates.

    PubMed

    Pacheco, Neith; Trombotto, Stphane; David, Laurent; Shirai, Keiko

    2013-07-01

    Production of chitin deacetylases from the phytopathogenic fungus Colletotrichum gloeosporioides was successfully achieved by submerged fermentation. The highest specific activity of 0.018 U mg(-1) of protein was obtained after 96 h of cultivation at pH 6 and 28C. Two bands with molecular weights of 35 kDa and 170 kDa determined with SDS-PAGE displayed deacetylase activities as detected in the zymograms. Reacetylated commercial chitosan (52% acetylation degree) was used as substrate for the extracellular crude extract in order to estimate the kinetic parameters of acetate production as undirected deacetylation measurement. The highest acetate production of 12.8 ?mol mL(-1) was obtained using 7.5 mg mL(-1) of substrate. The produced enzyme from C. gloeosporioides achieved up to 25% deacetylation of a chitin substrate (hydrolyzed biological chitin) having 80% degree of acetylation, MW of 10210(3) g mol(-1) and a crystallinity index of ca. 60%. PMID:23688474

  17. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    SciTech Connect

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and mineralisation produced by GSK-3 inhibition. • In rats, 3 GSK-3 inhibitors produced a unique serum bone turnover biomarker profile. • Enhanced bone formation was seen within 7 to 14 days of compound treatment in rats.

  18. Utilisation of chitinous materials in pigment adsorption.

    PubMed

    Wang, San-Lang; Chen, Yan-Cheng; Yen, Yue-Horng; Liang, Tzu-Wen

    2012-12-01

    The effect of adding the cells of four lactobacilli to a squid pen powder (SPP)-containing medium on prodigiosin (PG) production by Serratia marcescens TKU011 is examined. The best increase in PG productivity was shown by strain TKU012. Among the samples of strain TKU012 and the chitinous materials of cicada casting powder (CCP), shrimp shell powder (SSP), squid pen powder (SPP), ?-chitin, and ?-chitin, TKU012 cells displayed the best adsorption rate (84%) for PG, followed by CCP, SSP, SPP, ?-chitin, and ?-chitin. As for the water-soluble food colourants, Allura Red AC (R40) and Tartrazne (Y4), SPP and SSP had better adsorptive powers than pure chitin preparations, strain TKU012, and CCP. Treatment with organic solvents, hot alkali, or proteases (papain, bromelain) diminished the adsorption rates of the biosorbents. PMID:22953835

  19. Attenuation of Acute Nitrogen Mustard-Induced Lung Injury, Inflammation and Fibrogenesis by a Nitric Oxide Synthase Inhibitor

    PubMed Central

    Malaviya, Rama; Venosa, Alessandro; Hall, LeRoy; Gow, Andrew J.; Sinko, Patrick J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-01-01

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d - 28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS+ and cyclooxygenase-2+) and alternatively activated profibrotic (YM-1+ and galectin-3+) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2x/day, 1 d - 3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. PMID:22981630

  20. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    SciTech Connect

    Malaviya, Rama; Venosa, Alessandro; Hall, LeRoy; Gow, Andrew J.; Sinko, Patrick J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute lung injury induced by NM.

  1. Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors

    PubMed Central

    Duhoux, Arnaud; Délye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

  2. Long-term treatment with novel glycogen synthase kinase-3 inhibitor improves glucose homeostasis in ob/ob mice: molecular characterization in liver and muscle.

    PubMed

    Kaidanovich-Beilin, Oksana; Eldar-Finkelman, Hagit

    2006-01-01

    Glycogen synthase kinase-3 (GSK-3) is critically involved in insulin signaling, and its selective inhibition may present a new therapy for treatment of insulin resistance and type 2 diabetes. The current studies were designed to examine the impact of long-term in vivo inhibition of GSK-3 and its effects in the specific tissues. ob/ob mice were treated daily with one dose (400 nmol, i.p.) of a selective GSK-3 peptide inhibitor, L803-mts, for 3 weeks. Treatment with L803-mts reduced blood glucose levels, improved glucose tolerance, and prevented elevation of hyperglycemia with age. However, L803-mts did not affect either body weight or food consumption and was not toxic, as judged by histopathology and blood chemistry analyses. Consistent with these results, L803-mts suppressed mRNA levels of hepatic phosphoenolpyruvate carboxykinase (PEPCK) (50%) and increased hepatic glycogen content by 50%. On the other hand, L803-mts did not affect glucose 6-phosphate (G-6-P) phosphatase (G-6-Pase) mRNA levels or its enzymatic activity in the liver. Investigation for possible mechanisms responsible for PEPCK suppression indicated that phosphorylation of cAMP-responsive element transcription factor (CREB) at Ser(133) was reduced remarkably by L803-mts, which was also associated with reduced phosphorylation at Ser(129) and no change in total CREB. This suggested that PEPCK was suppressed by GSK-3 inhibition-mediated inactivation of CREB. In skeletal muscle, treatment with L803-mts led both to up-regulation in GLUT4 expression and to a 20% increase in glycogen content. Our studies show that long-term treatment with GSK-3 inhibitor improves glucose homeostasis in ob/ob mice and demonstrates a novel role of GSK-3 in regulating hepatic CREB activity and expression of muscle GLUT4. PMID:16169938

  3. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.

    PubMed Central

    Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson, W.; Aherne, G. W.; Hardcastle, A.; Boyle, F. T.

    1995-01-01

    Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation. PMID:7537518

  4. Reference genes to study herbicide stress response in Lolium sp.: up-regulation of P450 genes in plants resistant to acetolactate-synthase inhibitors.

    PubMed

    Duhoux, Arnaud; Délye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

  5. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule. PMID:25064440

  6. Combination of chiral linkers with thiophenecarboximidamide heads to improve the selectivity of inhibitors of neuronal nitric oxide synthase.

    PubMed

    Jing, Qing; Li, Huiying; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2014-09-15

    To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral linkers that derive from natural amino acid derivatives have been designed and synthesized. The new structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy synthesis and good inhibitory properties. Inhibitor (S)-9b exhibits a potency of 14.7 nM against nNOS and is 1134 and 322-fold more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 9b and propionate A on the heme and tetrahydrobiopterin (H4B) in nNOS, but not eNOS, contributes to its high selectivity. This work demonstrates the advantage of integrating known structures into structure optimization, and it should be possible to more readily develop compounds that incorporate bioavailability with these advanced features. Moreover, this integrative strategy is a general approach in new drug discovery. PMID:25149509

  7. Combination of Chiral Linkers with Thiophenecarboximidamide Heads to Improve the Selectivity of Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Jing, Qing; Li, Huiying; Roman, Linda J.; Martásek, Pavel; Poulos, Thomas L.; Silverman, Richard B.

    2014-01-01

    To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral linkers that derive from natural amino acid derivatives have been designed and synthesized. The new structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy synthesis and good inhibitory properties. Inhibitor (S)-9b exhibits a potency of 14.7 nM against nNOS and is 1134 and 322-fold more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 9b and propionate A on the heme and tetrahydrobiopterin (H4B) in nNOS, but not eNOS, contributes to its high selectivity. This work demonstrates the advantage of integrating known structures into structure optimization, and it should be possible to more readily develop compounds that incorporate bioavailability with these advanced features. Moreover, this integrative strategy is a general approach in new drug discovery. PMID:25149509

  8. Chitin, Chitinase Responses, and Invasive Fungal Infections

    PubMed Central

    Vega, Karina; Kalkum, Markus

    2012-01-01

    The human immune system is capable of recognizing and degrading chitin, an important cell wall component of pathogenic fungi. In the context of host-immune responses to fungal infections, herein we review the particular contributions and interplay of fungus and chitin recognition, and chitin-degrading enzymes, known as chitinases. The mechanisms of host chitinase responses may have implications for diagnostic assays as well as novel therapeutic approaches for patients that are at risk of contracting fatal fungal infections. PMID:22187561

  9. Design, Synthesis, Calorimetry and Crystallographic analysis of 2-Alkylaminoethyl-1,1-Bisphosphonates as inhibitors of Trypanosoma cruzi Farnesyl Diphosphate Synthase

    PubMed Central

    Aripirala, Srinivas; Szajnman, Sergio H.; Jakoncic, Jean; Rodriguez, Juan B.; Docampo, Roberto; Gabelli, Sandra B.; Amzel, L. Mario

    2016-01-01

    Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi--the etiologic agent of American trypanosomiasis (Chagas disease)--exhibiting IC50 values in the nanomolar range against the parasites. This activity is associated with inhibition at the low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). X-ray structures and thermodynamic data of the complexes TcFPPS with five compounds of this family show that the inhibitors bind to the allylic site of the enzyme with their alkyl chain occupying the cavity that binds the isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy compensated by a favorable entropy that results from a delicate balance between two opposing effects: the loss of conformational entropy due to freezing of single bond rotations, and the favorable burial of the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds and methyl branches should increase affinity substantially. PMID:22715997

  10. Modulation of thymidilate synthase and p53 expression by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with 5FU or raltitrexed.

    PubMed

    Di Gennaro, Elena; Bruzzese, Francesca; Pepe, Stefano; Leone, Alessandra; Delrio, Paolo; Subbarayan, Pochi R; Avallone, Antonio; Budillon, Alfredo

    2009-05-01

    Despite the introduction of several novel anticancer agents almost 50% of colorectal cancer (CRC) patients die for cancer suggesting the necessity of new therapeutical approaches. In this study we demonstrated that the HDAC inhibitor vorinostat exerted potent antiproliferative effect in a panel of mut- and wt-p53 human CRC cell lines. Moreover, in combination with 5-fluorouracil modulated by folinic acid (5FU-FA) or with Raltitrexed (RTX), both commonly used in the treatment of this disease, it showed a clear schedule-dependent synergistic antiproliferative interaction as demonstrated by calculating combination indexes. Only simultaneous, or 24 h pretreatment with vorinostat followed by either agent, produced synergistic effect paralleled by evident cell cycle perturbations with major S-phase arrest. Moreover, we provided for the first time evidences that vorinostat can overcome resistance to both 5FU and RTX. Downmodulation of Thymidilate synthase (TS) protein induced by vorinostat within 24 h, represented a key factor in enhancing the effects of both drugs in sensitive as well as resistant tumor cells. Furthermore, p53, whose wild-type expression is critical for sensitivity to 5FU and RTX, was upregulated by vorinostat in wt- and downregulated in mut-p53 cells, suggesting an additional mechanism of the antiproliferative synergistic interactions observed. Overall these data add new insights in the mechanism of vorinostat antitumor effect and suggested that the association of vorinostat plus 5FU-FA and/or RTX should be clinically explored. PMID:19270508

  11. Differentiations of chitin content and surface morphologies of chitins extracted from male and female grasshopper species.

    PubMed

    Kaya, Murat; Leleius, Evaldas; Nagrockait?, Radvil?; Sargin, Idris; Arslan, Gulsin; Mol, Abbas; Baran, Talat; Can, Esra; Bitim, Betul

    2015-01-01

    In this study, we used Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), and scanning electron microscopy (SEM) to investigate chitin structure isolated from both sexes of four grasshopper species. FT-IR, EA, XRD, and TGA showed that the chitin was in the alpha form. With respect to gender, two main differences were observed. First, we observed that the quantity of chitin was greater in males than in females and the dry weight of chitin between species ranged from 4.71% to 11.84%. Second, using SEM, we observed that the male chitin surface structure contained 25-90 nm wide nanofibers and 90-250 nm nanopores, while no pores or nanofibers were observed in the chitin surface structure of the majority of females (nanofibers were observed only in M. desertus females). In contrast, the elemental analysis, thermal properties, and crystalline index values for chitin were similar in males and females. Also, we carried out enzymatic digestion of the isolated chitins using commercial chitinase from Streptomyces griseus. We observed that there were no big differences in digestion rate of the chitins from both sexes and commercial chitin. The digestion rates were for grasshoppers' chitins; 88.45-95.48% and for commercial chitin; 94.95%. PMID:25635814

  12. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin.

    PubMed

    Svitil, A L; Chadhain, S; Moore, J A; Kirchman, D L

    1997-02-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products. PMID:16535505

  13. Differentiations of Chitin Content and Surface Morphologies of Chitins Extracted from Male and Female Grasshopper Species

    PubMed Central

    Kaya, Murat; Leleius, Evaldas; Nagrockait?, Radvil?; Sargin, Idris; Arslan, Gulsin; Mol, Abbas; Baran, Talat; Can, Esra; Bitim, Betul

    2015-01-01

    In this study, we used Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), and scanning electron microscopy (SEM) to investigate chitin structure isolated from both sexes of four grasshopper species. FT-IR, EA, XRD, and TGA showed that the chitin was in the alpha form. With respect to gender, two main differences were observed. First, we observed that the quantity of chitin was greater in males than in females and the dry weight of chitin between species ranged from 4.71% to 11.84%. Second, using SEM, we observed that the male chitin surface structure contained 25 90nm wide nanofibers and 90 250 nm nanopores, while no pores or nanofibers were observed in the chitin surface structure of the majority of females (nanofibers were observed only in M. desertus females). In contrast, the elemental analysis, thermal properties, and crystalline index values for chitin were similar in males and females. Also, we carried out enzymatic digestion of the isolated chitins using commercial chitinase from Streptomyces griseus. We observed that there were no big differences in digestion rate of the chitins from both sexes and commercial chitin. The digestion rates were for grasshoppers chitins; 88.4595.48% and for commercial chitin; 94.95%. PMID:25635814

  14. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10M)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0h, respectively, and increased 3NT and GSNO at 1.5-3.0h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

  15. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  16. Chitin Nanofiber Elucidates the Elicitor Activity of Polymeric Chitin in Plants

    PubMed Central

    Egusa, Mayumi; Matsui, Hidenori; Urakami, Takeshi; Okuda, Sanami; Ifuku, Shinsuke; Nakagami, Hirofumi; Kaminaka, Hironori

    2015-01-01

    Chitin, an N-acetyl-D-glucosamine polymer, is a component of fungal cell walls and a microbe/pathogen-associated molecular pattern that elicits plant defense responses. As polymeric chitin is difficult to handle due to its insolubility in water, many studies on chitin-induced immune responses have used water-soluble low-molecular weight chitin instead. Thus, it is unclear if polymeric chitin can induce resistance. Here, we examined the elicitor activity of chitin nanofiber (CNF) of submicron thickness prepared from polymeric chitin. CNF showed a high dispersing ability in water and induced both reactive oxygen species (ROS) production and chitin-induced defense-related gene expression in Arabidopsis thaliana seedlings. The Arabidopsis chitin elicitor receptor kinase 1 (Atcerk1) mutant, which is impaired in chitin perception, also failed to respond to CNF. CNF exposure triggered ROS generation in suspension-cultured cells from Oryza sativa. Furthermore, pre-treatment of Arabidopsis leaves with CNF effectively reduced pathogen infection by both the fungus Alternaria brassicicola and the bacterium Pseudomonas syringae pv. tomato DC3000. These results demonstrate that CNF has elicitor activity and will help define the role of polymeric chitin in plant immune responses. PMID:26697049

  17. Effects of S-ethylisothiourea, a potent inhibitor of nitric oxide synthase, alone or in combination with a nitric oxide donor in splanchnic artery occlusion shock.

    PubMed Central

    Squadrito, F.; Altavilla, D.; Squadrito, G.; Campo, G. M.; Ioculano, M.; Canale, P.; Rossi, F.; Saitta, A.; Caputi, A. P.

    1996-01-01

    1. The aim of this study was to compare the effects of an intravenous infusion of a potent and non selective nitric oxide synthase inhibitor S-ethylisothiourea (Ethyl-TU) with that of a nitric oxide (NO) donor on the pathological sequelae associated with splanchnic artery occlusion (SAO) shock. In addition the effects of the combination of these two treatments were also investigated. 2. SAO shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham operated animals were used as controls. Survival time, white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to evaluate neutrophil accumulation) and the responsiveness of aortic rings to acetylcholine (ACh, 10 nM-10 microM) and to phenylephrine (PE, 1 nM-10 microM) were studied. 3. SAO shocked rats had a decreased survival rate (0% survival 2 h after the release of occlusion) and survival time (76 +/- 10 min), increased MPO activity in the ileum (3.39 +/- 0.8 u x 10(-3) g-1 tissue), a marked leukopenia and a profound hypotension. In addition aortic rings from shocked rats showed a marked hyporeactivity to PE and reduced responsiveness to ACh. Endothelium denuded aortic rings had also a marked hyporeactivity to PE. 4. In vivo administration of Ethyl-TU (0.1 mg kg-1 h-1, beginning 1 min after the onset of reperfusion) significantly increased survival time and rate, improved mean arterial blood pressure, restored the responsiveness to PE, but did not change MPO activity, leukopenia or the impairment in the responsiveness of aortic rings to ACh. Addition of Ethyl-TU (2 microM) to endothelium denuded aortic rings in vitro, restored the marked hyporeactivity to PE. Administration of the NO donor C87-3754 (0.75 mg kg-1 h-1, beginning 1 min after the onset of reperfusion) slightly increased survival time and reduced MPO activity and leukopenia, but did not change survival rate and mean arterial blood pressure. In addition C87-3754 restored the responsiveness of aortic rings to ACh to control values, but did not modify the hyporeactivity to PE. The combination of these two interventions produced a higher degree of protection than either Ethyl-TU or C87-3754 alone. In fact, co-administration of Ethyl-TU plus C87-3754 completely prevented mortality, reduced MPO activity, attenuated leukopenia and the profound hypotension and restored the impaired responsiveness of aortic rings to PE and ACh. 5. Our study suggests that treatment with a nitric oxide synthase inhibitor combined with an NO donor may be a new therapeutic approach to the treatment of splanchnic artery occlusion shock. PMID:8872352

  18. Pharmacodynamic target evaluation of a novel oral glucan synthase inhibitor, SCY-078 (MK-3118), using an in vivo murine invasive candidiasis model.

    PubMed

    Lepak, Alexander J; Marchillo, Karen; Andes, David R

    2015-02-01

    Echinocandins inhibit the synthesis of ?-1,3-D-glucan in Candida and are the first-line therapy in numerous clinical settings. Their use is limited by poor oral bioavailability, and they are available only as intravenous therapies. Derivatives of enfumafungin are novel orally bioavailable glucan synthase inhibitors. We performed an in vivo pharmacodynamic (PD) evaluation with a novel enfumafungin derivative, SCY-078 (formerly MK-3118), in a well-established neutropenic murine model of invasive candidiasis against C. albicans, C. glabrata, and C. parapsilosis. The SCY-078 MICs varied 8-fold. Oral doses of 3.125 to 200 mg/kg SCY-078 salt in sterile water produced peak levels of 0.04 to 2.66 ?g/ml, elimination half-lives of 5.8 to 8.5 h, areas under the concentration-time curve from 0 to 24 h (AUC0-24 h) of 0.61 to 41.10 ?gh/ml, and AUC from 0 to infinity (AUC0-?) values of 0.68 to 40.31 ?gh/ml. The pharmacokinetics (PK) were approximately linear over the dose range studied. Maximum response (Emax) and PK/PD target identification studies were performed with 4 C. albicans, 4 C. glabrata, and 3 C. parapsilosis isolates. The PD index AUC/MIC was explored by using total (tAUC) and free (fAUC) drug concentrations. The maximum responses were 4.0, 4.0, and 4.3 log10 CFU/kidney reductions for C. albicans, C. glabrata, and C. parapsilosis, respectively. The AUC/MIC was a robust predictor of efficacy (R2, 0.53 to 0.91). The 24-h PD targets were a static dose of 63.5 mg/kg, a tAUC/MIC of 500, and an fAUC/MIC of 1.0 for C. albicans; a static dose of 58.4 mg/kg, a tAUC/MIC of 315, and an fAUC/MIC of 0.63 for C. glabrata; and a static dose of 84.4 mg/kg, a tAUC/MIC of 198, and an fAUC/MIC of 0.40 for C. parapsilosis. The mean fAUC/MIC values associated with a 1-log kill endpoint against these species were 1.42, 1.26, and 0.91 for C. albicans, C. glabrata, and C. parapsilosis, respectively. The static and 1-log kill endpoints were measured relative to the burden at the start of therapy. The static and 1-log kill doses, as well as the total and free drug AUC/MIC PD targets, were not statistically different between species but were numerically lower than those observed for echinocandins. SCY-078 is a promising novel oral glucan synthase inhibitor against Candida species, and further investigation is warranted. PMID:25512406

  19. Chitin-natural clay nanotubes hybrid hydrogel.

    PubMed

    Liu, Mingxian; Zhang, Yun; Li, Jingjing; Zhou, Changren

    2013-07-01

    Novel hybrid hydrogel was synthesized from chitin NaOH/urea aqueous solution in presence of halloysite nanotubes (HNTs) via crosslinking with epichlorohydrin. Fourier transform infrared (FT-IR) spectra and atomic force microscopy (AFM) results confirmed the interfacial interactions in the chitin-HNTs hybrid hydrogel. The compressive strength and shear modulus of chitin hydrogel were significantly increased by HNTs as shown in the static compressive experiment and rheology measurement. The hybrid hydrogels showed highly porous microstructures by scanning electron microscopy (SEM). The swelling ratio of chitin hydrogel decreased because of the addition of HNTs. The malachite green's absorption experiment result showed that the hybrid hydrogel exhibited much higher absorption rate than the pure chitin hydrogel. The prepared hybrid hydrogel had potential applications in waste treatment and biomedical areas. PMID:23535366

  20. Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures?

    PubMed Central

    Walker, Claire A.; Gmez, Beatriz L.; Mora-Montes, Hctor M.; Mackenzie, Kevin S.; Munro, Carol A.; Brown, Alistair J. P.; Gow, Neil A. R.; Kibbler, Christopher C.; Odds, Frank C.

    2010-01-01

    The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3? and chs2? chs3? mutants but were fully externalized in chs8? and chs2? chs8? mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization. PMID:20543065

  1. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis

    PubMed Central

    Lo, Hui-Chen; Hung, Ching-Yi; Huang, Fu-Huan; Su, Tzu-Cheng; Lee, Chien-Hsing

    2016-01-01

    The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats. PMID:27007815

  2. Nuclear Factor-?B (NF-?B) Mediates a Protective Response in Cancer Cells Treated with Inhibitors of Fatty Acid Synthase*

    PubMed Central

    Lemmon, Colleen R. M.; Woo, Ju-Hyung; Tully, Ellen; Wilsbach, Kathleen; Gabrielson, Edward

    2011-01-01

    The efficacy of drugs used to treat cancer can be significantly attenuated by adaptive responses of neoplastic cells to drug-induced stress. To determine how cancer cells respond to inhibition of the enzyme fatty acid synthase (FAS), we focused on NF-?B-mediated pathways, which can be activated by various cellular stresses. Treating lung cancer cells with C93, a pharmacological inhibitor of FAS, results in changes indicative of a rapid initiation of NF-?B signaling, including translocation of RelA/p65 NF-?B to the nucleus, activation of a transfected NF-?B-luciferase reporter, and increased expression of NF-?B-dependent transcripts, IL-6, IL-8, and COX-2. Verifying that these responses to C93 are specifically related to inhibition of FAS, we confirmed that levels of these same transcripts increase in response to siRNA targeting FAS. Inhibiting this NF-?B response (either by transfecting a mutant I?B? or treating with bortezomib) resulted in increased cell killing by C93, indicating that the NF-?B response is protective in this setting. Because inhibiting FAS leads to accumulation of intermediate metabolites of fatty acid biosynthesis, we then questioned whether protein kinase C (PKC) is involved in this response to metabolic stress. Immunofluorescence microscopy revealed that C93 treatment results in cellular translocation of PKC? and PKC? isoforms and increased PKC?-dependent phosphorylation of the I?B? subunit of NF-?B. Furthermore, inhibiting PKC activity with RO-318220 or PKC? isoform-specific siRNA attenuates C93-induced I?B? phosphorylation and NF-?B activation and also potentiates C93-induced cell killing. These results suggest a link between PKC and NF-?B in protecting cancer cells from metabolic stress induced by inhibiting FAS. PMID:21768098

  3. A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

    PubMed Central

    Woodard, Crystal; Liao, Gangling; Goodwin, C. Rory; Hu, Jianfei; Xie, Zhi; dos Reis, Thaila F.; Newman, Rob; Rho, Heesool; Qian, Jiang

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells. PMID:26109723

  4. Separation of C-glycoside flavonoids from Aleurites moluccana using chitin and full N-acetylated chitin.

    PubMed

    Morsch, Michele; Girardi, Leury G J; Cechinel-Filho, Valdir; Meyre-Silva, Christiane; Rodrigues, Clvis Antonio

    2002-01-01

    This paper describes a comparative study using different chromatographic supports (fully N-acetylated chitin, chitin and silica gel) to separate the flavonoids swertisin and 2"-O-rhamnosylswertisin from Aleurites moluccana. The results show that the flavonoids have apparently been separated by the hydrogen bond between the stationary phase (chitin and chitin-100) and flavonoids under the conditions studied. PMID:12440742

  5. Effect of Polyoxin D on Chitin Synthesis and Septum Formation in Saccharomyces cerevisiae

    PubMed Central

    Bowers, Blair; Levin, Gary; Cabib, Enrico

    1974-01-01

    The normal sequence of cell separation in Saccharomyces cerevisiae begins with the formation of a primary septum, presumably consisting of chitin, on which secondary septa are later deposited. In the presence of the antibiotic polyoxin D, a potent inhibitor of chitin synthetase, pairs of abnormal cells of two different types were observed by phase-contrast microscopy: the “exploded pair,” consisting of two lysed cells from which the cytoplasm had been extruded at the cell junction, and the “refringent pair,” consisting of two highly refractile cells joined by a thin bridge. Thus, in both cases the septal region appears to be affected. Observations with the electron microscope showed that the primary chitin septum was not formed in either of these cell types, and as a consequence secondary septa of varying thicknesses were laid down in an abnormal pattern. With [3H]glucose as carbon source the incorporation of tritium into the chitin of abnormal cells was inhibited about 90%, whereas the labeling of mannan was normal and that of glucan somewhat reduced. The effective concentrations of polyoxin D (0.1 to 1 mg/ml) were much greater than those required to inhibit chitin synthesis in vitro. Dimethylsulfoxide and amphotericin B, both known to increase cell permeability, enhanced the action of the antibiotic. Images PMID:4604393

  6. Direct compression properties of chitin and chitosan.

    PubMed

    Mir, Viviana Garca; Heinmki, Jyrki; Antikainen, Osmo; Revoredo, Ofelia Bilbao; Colarte, Antonio Iraizoz; Nieto, Olga Maria; Yliruusi, Jouko

    2008-08-01

    Deformation and compaction properties of native amino poly-saccharides chitin and chitosan were studied and compared with those obtained with established pharmaceutical direct compression excipients. An instrumented single-punch tablet machine was used for tablet compaction. The following compression parameters were evaluated: a ratio of crushing strength and compression pressure, plasticity and elasticity factor (PF and EF), tensile strength and R-value. Chitin and chitosan were found to have a marked tendency to plastic deformation, and both showed a good compression behaviour compared with the other direct compression excipients including microcrystalline cellulose. It is concluded that chitin and chitosan are potential co-excipients for direct compression applications. PMID:18406116

  7. In situ microscopic observation of chitin and fungal cells with chitinous cell walls in hydrothermal conditions

    PubMed Central

    Deguchi, Shigeru; Tsujii, Kaoru; Horikoshi, Koki

    2015-01-01

    Recent findings of intact chitin in fossil records suggest surprisingly high recalcitrance of this biopolymer during hydrothermal treatments. We also know in the experience of everyday life that mushroom, cells of which have chitinous cell walls, do not fall apart however long they are simmered. We used in situ optical microscopy to examine chitin and fungal cells with chitinous cell walls during hydrothermal treatments, and obtained direct evidence that they remained undegraded at temperatures well over 200?C. The results show very hot and compressed water is needed to make mushrooms mushy. PMID:26148792

  8. Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

    SciTech Connect

    Wu Defeng; Cederbaum, Arthur . E-mail: arthur.cederbaum@mssm.edu

    2006-10-15

    Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N {sup G}-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 {+-} 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 {+-} 5%, while, SNAP or DETA-NONO increased viability to 66 {+-} 8 or 71 {+-} 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.

  9. Spermine synthase

    PubMed Central

    Michael, Anthony J.

    2010-01-01

    Spermine is present in many organisms including animals, plants, some fungi, some archaea, and some bacteria. It is synthesized by spermine synthase, a highly specific aminopropyltransferase. This review describes spermine synthase structure, genetics, and function. Structural and biochemical studies reveal that human spermine synthase is an obligate dimer. Each monomer contains a C-terminal domain where the active site is located, a central linking domain that also forms the lid of the catalytic domain, and an N-terminal domain that is structurally very similar to S-adenosylmethionine decarboxylase. Gyro mice, which have an X-chromosomal deletion including the spermine synthase (SMS) gene, lack all spermine and have a greatly reduced size, sterility, deafness, neurological abnormalities, and a tendency to sudden death. Mutations in the human SMS lead to a rise in spermidine and reduction of spermine causing Snyder-Robinson syndrome, an X-linked recessive condition characterized by mental retardation, skeletal defects, hypotonia, and movement disorders. PMID:19859664

  10. Chitin enhances obese inflammation ex vivo.

    PubMed

    Huang, Chun-Jung; Beasley, Kathleen N; Acevedo, Edmund O; Franco, Robert L; Jones, Tamekia L; Mari, David C; Shibata, Yoshimi

    2014-01-01

    Infection has been implicated as a co-risk factor for obesity, but the mechanism remains uncertain. Elevated levels of plasma chitinase 3-like 1 (CHI3L1) are found in obese individuals. Since CHI3L1 is produced by activated immune cells including macrophages and recognizes microbial N-acetylglucosamine polymer (chitin), we asked whether the plasma CHI3L1 protein change in obese individuals might alter their innate immune response to chitin. Thirty-six subjects (15 obese and 21 non-obese), ages 18-30 years, were recruited. Peripheral blood mononuclear cells (PBMCs) were cultured with chitin microparticles (CMP; 1-10 μm) for 24h; tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and CHI3L1 in the culture supernatants were measured. We chose CMP, since neither large chitin beads (40-100 μm), chitosan microparticles (1-10 μm), nor soluble chitin induced the cytokine/CHI3L1 production by PBMCs isolated from non-obese PBMCs ex vivo. We found that the quantity of IL-6, but not TNF-α or CHI3L1, induced by CMP was significantly correlated with plasma IL-6, BMI, waist/hip circumferences, fasting plasma insulin, and insulin resistance. These findings suggest that chitin, a substrate of CHI3L1, further promotes obese inflammation in a size- and chemical composition- dependent manner. PMID:24055693

  11. A phase I and pharmacokinetic study of 1843U89, a noncompetitive inhibitor of thymidylate synthase, in patients with advanced solid malignancies.

    PubMed

    Schwartz, G; Johnson, T R; Goetz, A; Burris, H; Smetzer, L; Lampkin, T; Sailstad, J; Hohneker, J A; Von Hoff, D D; Rowinsky, E K

    2001-07-01

    This study was performed to assess the feasibility of administering 1843U89, a potent, noncompetitive inhibitor of thymidylate synthase that does not require polyglutamation for activity, as a 2-min i.v. infusion daily for 5 days every 3 weeks, to determine whether folic acid supplementation ameliorates the toxic effects of 1843U89 and permits further dose escalation, and to recommend doses of 1843U89 administered without and with folic acid for further clinical evaluations. The study also sought to characterize the pharmacokinetic behavior of 1843U89 and to seek preliminary evidence of anticancer activity. Patients with advanced solid malignancies were treated with escalating doses of 1843U89 as a 2-min i.v. infusion daily for 5 days every 3 weeks. Initially, patients were treated in the absence of high-dose folic acid until dose-limiting toxicity was consistently noted. Next, patients were treated with escalating doses of 1843U89 preceded by 1000 mg of folic acid administered p.o. 30 min before each of the 5 daily doses of 1843U89. Patients (32) received 101 total courses of 1843U89 at doses ranging from 1 to 6 mg/m(2)/day with and without folic acid. At the 2 mg/m(2)/day dose level without folic acid, 2 of 7 new patients experienced dose-limiting toxicity, principally neutropenia, mucositis, and malaise in 3 of 11 courses. 1843U89 doses were further increased with folic acid to 6 mg/m(2)/day, but repetitive treatment was not feasible at this dose level because of an unacceptable high incidence of severe neutropenia and mucositis. Other toxicities included thrombocytopenia, rash, and fever. In contrast, repetitive treatment at the 5 mg/m(2)/day dose level was feasible. The pharmacokinetics of 1843U89 were neither dose dependent nor affected by folic acid. On day 1, clearance, terminal half-life, and steady-state volume of distribution values averaged 47.1 +/- 21.7 ml/min/m(2), 7.72 +/- 4.09 h, and 16.7 +/- 8.8 liter/m(2)/h, respectively. The results of the study indicate that the administration of 1843U89 as a 2-min infusion daily for 5 days every 3 weeks without and with folic acid is feasible at 1843U89 doses as high as 2 and 5 mg/m(2)/day, respectively. Because folic acid pretreatment results in no diminution of the antitumor activity of 1843U89 in preclinical studies and ameliorates the toxic effects of 1843U89 in both preclinical models and cancer patients, the therapeutic index of 1843U89 may be enhanced by folic acid pretreatment and, therefore, the development of 1843U89 with folic acid is warranted. However, the question of whether to administer 1843U89 at a dose of 2 mg/m(2)/day with folic acid, which is associated with negligible toxicity, or at its highest feasible dose with folic acid, 5 mg/m(2)/day, should be addressed in appropriately designed trials. PMID:11448903

  12. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, interacts with gastric oxidative metabolism and enhances stress-induced gastric lesions.

    PubMed

    Kwiecien, S; Ptak-Belowska, A; Krzysiek-Maczka, G; Targosz, A; Jasnos, K; Magierowski, M; Szczyrk, U; Brzozowski, B; Konturek, S J; Konturek, P C; Brzozowski, T

    2012-10-01

    Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase known to exert vasoconstriction of vascular bed. The elevation of ADMA has been considered as the cardiovascular risk factor associated with hyperlipidemia, hypercholesterolemia and metabolic syndrome. ADMA is produced by the action of dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Previous studies have shown that endogenous NO plays an important role in the mechanism of gastric mucosal defense, but the role of ADMA in the pathogenesis of serious clinical entity, such as the acute gastric mucosal injury induced by stress has been little studied. In present study, we determined the effect of intragastric (i.g.) pretreatment with ADMA applied in graded doses ranging from 0.1 up to 20 mg/kg on gastric mucosal lesions induced by 3.5 h of water immersion and restraint stress (WRS). The number of gastric lesions was determined by planimetry and the gastric blood flow (GBF) was assessed by laser Doppler technique. The malondialdehyde and 4-hydroxynonenal (MDA+4-HNE) concentration, as an index of oxygen radical-lipid peroxidation was assessed in the gastric mucosa in rats exposed to WRS with or without ADMA administration. Proinflammatory cytokines IL-1β, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1β and TNF-α were analyzed by RT-PCR and ELISA, respectively. The exposure of rats to WRS for 3.5 h produced acute gastric lesions accompanied by a significant rise in the plasma ADMA levels and a significant fall in the GBF, an increase in MDA+4-HNE concentrations and the significant increase in the expression and release of IL-1β and TNF-α. The pretreatment with ADMA, applied i.g. 30 min before WRS dose-dependently, aggravated WRS damage and this effect was accompanied by a further significant fall in the GBF. The ADMA induced exacerbation of WRS lesions and the accompanying rise in the plasma ADMA levels and the fall in GBF were significantly attenuated by concurrent treatment with glyceryl trinitrate (GTN) (10 mg/kg i.g.) in the presence of ADMA. Administration of ADMA resulted in a significant decrease in the expression of SOD and GPx mRNAs and the up-regulation of mRNA for IL-1β and TNF-α followed by an increase in these plasma cytokine levels as compared to respective values observed in vehicle-pretreated animals. We conclude that 1) ADMA could be implicated in the mechanism of WRS-induced ulcerogenesis, 2) ADMA exacerbates WRS-induced gastric lesions due to enhancement in neutrophil dependent lipid peroxidation and overexpression and release of proinflammatory cytokines IL-1β and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity. PMID:23211305

  13. Bacterial chitin degradation—mechanisms and ecophysiological strategies

    PubMed Central

    Beier, Sara; Bertilsson, Stefan

    2013-01-01

    Chitin is one the most abundant polymers in nature and interacts with both carbon and nitrogen cycles. Processes controlling chitin degradation are summarized in reviews published some 20 years ago, but the recent use of culture-independent molecular methods has led to a revised understanding of the ecology and biochemistry of this process and the organisms involved. This review summarizes different mechanisms and the principal steps involved in chitin degradation at a molecular level while also discussing the coupling of community composition to measured chitin hydrolysis activities and substrate uptake. Ecological consequences are then highlighted and discussed with a focus on the cross feeding associated with the different habitats that arise because of the need for extracellular hydrolysis of the chitin polymer prior to metabolic use. Principal environmental drivers of chitin degradation are identified which are likely to influence both community composition of chitin degrading bacteria and measured chitin hydrolysis activities. PMID:23785358

  14. Chitin accelerates activation of a novel haloarchaeal serine protease that deproteinizes chitin-containing biomass.

    PubMed

    Zhang, Yaoxin; Wang, Mengxin; Du, Xin; Tang, Wei; Zhang, Li; Li, Moran; Wang, Jian; Tang, Bing; Tang, Xiao-Feng

    2014-09-01

    The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I(WT)), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M(WT)) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-I(WT) to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I(WT) released soluble peptides from SSP faster than did M(WT). Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass. PMID:25002433

  15. Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

    PubMed Central

    Zhang, Yaoxin; Wang, Mengxin; Du, Xin; Tang, Wei; Zhang, Li; Li, Moran; Wang, Jian; Tang, Bing

    2014-01-01

    The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-IWT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (MWT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-IWT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-IWT released soluble peptides from SSP faster than did MWT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass. PMID:25002433

  16. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    SciTech Connect

    Horst, M.N. )

    1990-12-01

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.

  17. Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors.

    PubMed

    Shen, Yuefei; Zhang, Shiyi; Zhang, Fuwu; Loftis, Alexander; Pavía-Sanders, Adriana; Zou, Jiong; Fan, Jingwei; Taylor, John-Stephen A; Wooley, Karen L

    2013-10-18

    A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury. PMID:23999874

  18. Chitin induces natural competence in Vibrio cholerae.

    PubMed

    Meibom, Karin L; Blokesch, Melanie; Dolganov, Nadia A; Wu, Cheng-Yen; Schoolnik, Gary K

    2005-12-16

    The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress. PMID:16357262

  19. Dual Inhibitors of Thymidylate Synthase and Dihydrofolate Reductase as Antitumour Agents: Design, Synthesis and Biological Evaluation of Classical and Nonclassical Pyrrolo[2,3-d]pyrimidine Antifolates1

    PubMed Central

    Gangjee, Aleem; Jain, Hiteshkumar D.; Phan, Jaclyn; Lin, Xin; Song, Xiaohong; McGuire, John J.; Kisliuk, Roy L.

    2008-01-01

    We designed and synthesized a classical analog N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogs 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumour agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analog 4, the ester obtained from the reaction was deprotected and coupled with diethyl-L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC50 = 90 nM) and human DHFR (IC50 = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogs were only inhibitors of human TS with IC50 values of 0.23-26 ?M. PMID:16451071

  20. Design, Synthesis, and Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors

    PubMed Central

    Gangjee, Aleem; Li, Wei; Yang, Jie; Kisliuk, Roy L.

    2013-01-01

    We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-l-glutamic acid 4 and 11 nonclassical analogues 515 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-dimethylpropanamide, 29, to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from the N-benzylation reaction was deprotected and coupled with diethyl l-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC50 = 46 nM, about 206-fold more potent than pemetrexed) and DHFR (IC50 = 120 nM, about 55-fold more potent than pemetrexed). The nonclassical analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition along with >100-fold selectivity compared to human DHFR. PMID:18072727

  1. Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

    PubMed

    Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

    2015-11-25

    Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders. PMID:26486317

  2. Synthesis and physicochemical characterization of chitin dihexanoate - A new biocompatible chitin derivative - In comparison to chitin dibutyrate.

    PubMed

    Sko?ucka-Szary, Karolina; Rami?ga, Aleksandra; Piaskowska, Wanda; Janicki, Bartosz; Grala, Magdalena; Rieske, Piotr; Bartczak, Zbigniew; Piaskowski, Sylwester

    2016-03-01

    Chitin dihexanoate (DHCH) is the novel biocompatible and technologically friendly highly substituted chitin diester. Here we described optimization of DHCH and chitin dibutyrate (dibutyryl chitin, DBC) synthesis conditions (temperature and reaction time) to obtain desired polymers with high reaction yield, high substitution degree (close to 2) and appropriately high molecular weights. A two-step procedure, employing acidic anhydrides (hexanoic or butyric anhydride) as the acylation agent and methanesulfonic acid both as the catalyst and the reaction medium, was applied. Chemical structures of DBC and DHCH were confirmed by NMR ((1)H and (13)C) and IR investigations. Mechanical properties, thermogravimetric analysis, differential scanning calorimetry and biocompatibility (Neutral red uptake assay, Skin Sensitization and Irritation Tests) were assessed. Both polymers proved highly biocompatible (non-cytotoxic in vitro, non-irritating and non-allergic to skin) and soluble in several organic solvents (dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, ethanol and others). It is worth emphasizing that DHCH and DBC can be easily processed by solvent casting method and the salt-leaching method, what gives the opportunity to prepare highly porous structures, which can be further successfully applied as the material for wound dressings and scaffolds for tissue engineering. PMID:26706556

  3. 4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic Acid (C75), an Inhibitor of Fatty-acid Synthase, Suppresses the Mitochondrial Fatty Acid Synthesis Pathway and Impairs Mitochondrial Function*

    PubMed Central

    Chen, Cong; Han, Xiao; Zou, Xuan; Li, Yuan; Yang, Liang; Cao, Ke; Xu, Jie; Long, Jiangang; Liu, Jiankang; Feng, Zhihui

    2014-01-01

    4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial ?-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment. PMID:24784139

  4. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests, that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.

  5. Radiosynthesis and preliminary PET evaluation of glycogen synthase kinase 3β (GSK-3β) inhibitors containing [(11)C]methylsulfanyl, [(11)C]methylsulfinyl or [(11)C]methylsulfonyl groups.

    PubMed

    Kumata, Katsushi; Yui, Joji; Xie, Lin; Zhang, Yiding; Nengaki, Nobuki; Fujinaga, Masayuki; Yamasaki, Tomoteru; Shimoda, Yoko; Zhang, Ming-Rong

    2015-08-15

    Three compounds 1-3 containing methyl-sufanyl, sufinyl, or sulfonyl groups are strong inhibitors of glycogen synthase kinase 3β (GSK-3β), an enzyme associated with Alzheimer's disease. We labeled 1-3 with (11)C for a positron emission tomography (PET) brain imaging study. A novel thiophenol precursor 4 for radiosynthesis was prepared by reacting sulfoxide 2 with trifluoroacetic anhydride. [(11)C]1 was synthesized by reacting 4 with [(11)C]methyl iodide in 52 ± 5% radiochemical yield (n = 5, based on [(11)C]CO2, corrected for decay). Oxidation of [(11)C]1 with Oxone® produced [(11)C]2 and [(11)C]3, respectively. PET with [(11)C]1 and [(11)C]3 showed 2 fold higher brain uptake of radioactivity in a mouse model of cold water stress in which GSK-3β expression was increased, than in the controls. PMID:26067173

  6. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  7. Poriferan chitin as a template for hydrothermal zirconia deposition

    NASA Astrophysics Data System (ADS)

    Wysokowski, Marcin; Motylenko, Mykhaylo; Bazhenov, Vasilii V.; Stawski, Dawid; Petrenko, Iaroslav; Ehrlich, Andre; Behm, Thomas; Kljajic, Zoran; Stelling, Allison L.; Jesionowski, Teofil; Ehrlich, Hermann

    2013-09-01

    Chitin is a thermostable biopolymer found in various inorganic-organic skeletal structures of numerous invertebrates including sponges (Porifera). The occurrence of chitin within calcium- and silica-based biominerals in organisms living in extreme natural conditions has inspired development of new (extreme biomimetic) synthesis route of chitin-based hybrid materials in vitro. Here, we show for the first time that 3D-?-chitin scaffolds isolated from skeletons of the marine sponge Aplysina aerophoba can be effectively mineralized under hydrothermal conditions (150C) using ammonium zirconium(IV) carbonate as a precursor of zirconia. Obtained chitin-ZrO2 hybrid materials were characterized by FT-IR, SEM, HRTEM, as well as light and confocal laser microscopy. We suggest that formation of chitin-ZrO2 hybrids occurs due to hydrogen bonds between chitin and ZrO2.

  8. Stabilizing oil-in-water emulsions with regenerated chitin nanofibers.

    PubMed

    Zhang, Ying; Chen, Zhigang; Bian, Wenyang; Feng, Li; Wu, Zongwei; Wang, Peng; Zeng, Xiaoxiong; Wu, Tao

    2015-09-15

    Natural chitin is a highly crystalline biopolymer with poor aqueous solubility. Thus direct application of chitin is rather limited unless chemical modifications are made to improve its solubility in aqueous media. Through a simple dissolution and regeneration process, we have successfully prepared chitin nanofibers with diameters around 50nm, which form a stable suspension at concentrations higher than 0.50% and a self-supporting gel at concentrations higher than 1.00%. Additionally, these nanofibers can stabilize oil-in-water emulsions with oil fraction more than 0.50 at chitin usage level of 0.01g/g oil. The droplet sizes of the resulting emulsions decrease with increasing chitin concentrations and decreasing oil fraction. Confocal laser scanning micrographs demonstrate the adsorption of chitin nanofibers on the emulsion droplet surface, which indicates the emulsion stabilization is through a Pickering mechanism. Our findings allow the direct application of chitin in the food industry without chemical modifications. PMID:25863618

  9. Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis.

    PubMed

    Trudel, J; Asselin, A

    1990-09-01

    Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis. PMID:2281870

  10. Chitin: 'Forgotten' Source of Nitrogen: From Modern Chitin to Thermally Mature Kerogen: Lessons from Nitrogen Isotope Ratios

    USGS Publications Warehouse

    Schimmelmann, A.; Wintsch, R.P.; Lewan, M.D.; DeNiro, M.J.

    1998-01-01

    Chitinous biomass represents a major pool of organic nitrogen in living biota and is likely to have contributed some of the fossil organic nitrogen in kerogen. We review the nitrogen isotope biogeochemistry of chitin and present preliminary results suggesting interaction between kerogen and ammonium during thermal maturation. Modern arthropod chitin may shift its nitrogen isotope ratio by a few per mil depending on the chemical method of chitin preparation, mostly because N-containing non-amino-sugar components in chemically complex chitin cannot be removed quantitatively. Acid hydrolysis of chemically complex chitin and subsequent ion-chromatographic purification of the "deacetylated chitin-monomer" D-glucosamine (in hydrochloride form) provides a chemically well-defined, pure amino-sugar substrate for reproducible, high-precision determination of ??15N values in chitin. ??15N values of chitin exhibited a variability of about one per mil within an individual's exoskeleton. The nitrogen isotope ratio differed between old and new exoskeletons by up to 4 per mil. A strong dietary influence on the ??15N value of chitin is indicated by the observation of increasing ??15N values of chitin from marine crustaceans with increasing trophic level. Partial biodegradation of exoskeletons does not significantly influence ??15N values of remaining, chemically preserved amino sugar in chitin. Diagenesis and increasing thermal maturity of sedimentary organic matter, including chitin-derived nitrogen-rich moieties, result in humic compounds much different from chitin and may significantly change bulk ??15N values. Hydrous pyrolysis of immature source rocks at 330??C in contact with 15N-enriched NH4Cl, under conditions of artificial oil generation, demonstrates the abiogenic incorporation of inorganic nitrogen into carbon-bound nitrogen in kerogen. Not all organic nitrogen in natural, thermally mature kerogen is therefore necessarily derived from original organic matter, but may partly result from reaction with ammonium-containing pore waters.

  11. Esterification of beta-chitin via intercalation by carboxylic anhydrides.

    PubMed

    Yoshifuji, A; Noishiki, Y; Wada, M; Heux, L; Kuga, S

    2006-10-01

    beta-chitin is known to form intercalation complexes with aliphatic alcohols and amines. We found that it also forms complexes with carboxylic anhydrides. When the beta-chitin-acetic anhydride complex was heated to 105 degrees C, the hydroxyl groups of chitin were acetylated by a host-guest reaction, maintaining the host's crystal structure. Structures of complex and acetylated products were analyzed by X-ray diffraction, (13)C CP/MAS NMR, and infrared spectroscopy. The maximum degree of substitution (DS) was close to 1.0, suggesting regioselective esterification at the C6 position of chitin. Partially acetylated beta-chitin with a DS of 0.4 could incorporate various guest species that are difficult to be incorporated by original beta-chitin. In contrast, beta-chitin acetate with a DS of 1 lost the ability to form a complex. Intercalation complexes of beta-chitin with cyclic anhydrides (succinic and maleic) also underwent esterification by heating, and the products with a DS of approximately 1 dissolved in aqueous alkali, apparently as the result of the dissociation of introduced carboxyl groups. These phenomena are potentially useful in controlling the complexation ability of beta-chitin and the preparation of regioselectively esterified chitin derivatives. PMID:17025365

  12. Comparison of chitin structures isolated from seven Orthoptera species.

    PubMed

    Kaya, Murat; Erdogan, Sevil; Mol, Abbas; Baran, Talat

    2015-01-01

    Differences in the physichochemical properties of the chitin structure of the exoskeleton of seven species from four genera were investigated in this study. The same method was used to isolate the chitin structure of the seven species. The physicochemical properties of the isolated chitins were revealed by ESEM, FTIR, TGA and XRD analyses. The FTIR, TGA and XRD results from the chitin samples were similar. The surface morphologies of the chitins were investigated by ESEM and interesting results were noted. While the surface morphologies of the chitins isolated from two species within the same genus were quite different, the surface morphologies of chitins isolated from species belonging to different genera showed similarity. It was determined that the dry weight chitin contents of the grasshopper species varied between 5.3% and 8.9%. The results of molecular analysis showed that the chitins from seven Orthoptera species (between 5.2 and 6.8 kDa) have low molecular weights. Considering that these invasive and harmful species are killed with insecticides and go to waste in large amounts, this study suggests that they should be collected and evaluated as an alternative chitin source. PMID:25290985

  13. Conservation of the chitin utilization pathway in the Vibrionaceae.

    PubMed

    Hunt, Dana E; Gevers, Dirk; Vahora, Nisha M; Polz, Martin F

    2008-01-01

    Vibrionaceae are regarded as important marine chitin degraders, and attachment to chitin regulates important biological functions; yet, the degree of chitin pathway conservation in Vibrionaceae is unknown. Here, a core chitin degradation pathway is proposed based on comparison of 19 Vibrio and Photobacterium genomes with a detailed metabolic map assembled for V. cholerae from published biochemical, genomic, and transcriptomic results. Further, to assess whether chitin degradation is a conserved property of Vibrionaceae, a set of 54 strains from 32 taxa were tested for the ability to grow on various forms of chitin. All strains grew on N-acetylglucosamine (GlcNAc), the monomer of chitin. The majority of isolates grew on alpha (crab shell) and beta (squid pen) chitin and contained chitinase A (chiA) genes. chiA sequencing and phylogenetic analysis suggest that this gene is a good indicator of chitin metabolism but appears subject to horizontal gene transfer and duplication. Overall, chitin metabolism appears to be a core function of Vibrionaceae, but individual pathway components exhibit dynamic evolutionary histories. PMID:17933912

  14. Characterization of genes for chitin catabolism in Haloferax mediterranei.

    PubMed

    Hou, Jing; Han, Jing; Cai, Lei; Zhou, Jian; Lü, Yang; Jin, Cheng; Liu, Jingfang; Xiang, Hua

    2014-02-01

    Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion. PMID:23674154

  15. Design and Synthesis of 2-Amino-4-methylpyridine Analogues as Inhibitors for Inducible Nitric Oxide Synthase and in vivo Evaluation of [18F]6-(2-Fluoropropyl)-4-methyl-pyridin-2-amine as a Potential PET Tracer for Inducible Nitric Oxide Synthase

    PubMed Central

    Zhou, Dong; Lee, Hsiaoju; Rothfuss, Justin M.; Chen, Delphine L.; Ponde, Datta E.; Welch, Michael J.; Mach, Robert H.

    2009-01-01

    A series of position-6 substituted 2-amino-4-methylpyridine analogues was synthesized and compounds 9, 18, and 20 were identified as the inhibitors with the greatest potential to serve as PET tracers for imaging inducible nitric oxide synthase (iNOS). [18F]9 was synthesized and evaluated in a mouse model of lipopolysaccharide (LPS)-induced iNOS activation. In vivo biodistribution studies of [18F]9 indicate higher tracer uptake in the lungs of the LPS-treated mice when compared to control mice. Tracer uptake at 60 min post-injection was reduced in a blocking study using a known inhibitor of iNOS. The expression of iNOS was confirmed by Western blot analysis of lung samples from the LPS-treated mice. MicroPET studies also demonstrated accumulation of radiotracer in the lungs of the LPS-treated mice. Taken collectively, these data suggest that [18F]9 shows favorable properties as a PET tracer to image iNOS activation with PET. PMID:19323559

  16. Synthesis and enzyme inhibitory activity of the s-nucleoside analogue of the ribitylaminopyrimidine substrate of lumazine synthase and product of riboflavin synthase.

    PubMed

    Talukdar, Arindam; Illarionov, Boris; Bacher, Adelbert; Fischer, Markus; Cushman, Mark

    2007-09-14

    Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin. To obtain structural and mechanistic probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a sulfur analogue of the pyrimidine substrate of the lumazine synthase-catalyzed reaction and product of the riboflavin synthase-catalyzed reaction was designed. Facile syntheses of the S-nucleoside 5-amino-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione hydrochloride (15) and its nitro precursor 5-nitro-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione (14) are described. These compounds were tested against lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. Compounds 14 and 15 were found to be inhibitors of both riboflavin synthase and lumazine synthase. Compound 14 is an inhibitor of Bacillus subtilis lumazine synthase (Ki 26 microM), Schizosaccharomyces pombe lumazine synthase (Ki 2.0 microM), Mycobacterium tuberculosis lumazine synthase (Ki 11 microM), Escherichia coli riboflavin synthase (Ki 2.7 microM), and Mycobacterium tuberculosis riboflavin synthase (Ki 0.56 muM), while compound 15 is an inhibitor of B. subtilis lumazine synthase (Ki 2.6 microM), S. pombe lumazine synthase (Ki 0.16 microM), M. tuberculosis lumazine synthase (Ki 31 microM), E. coli riboflavin synthase (Ki 47 microM), and M. tuberculosis riboflavin synthase (Ki 2.5 microM). PMID:17696548

  17. Biosynthesis, Turnover, and Functions of Chitin in Insects.

    PubMed

    Zhu, Kun Yan; Merzendorfer, Hans; Zhang, Wenqing; Zhang, Jianzhen; Muthukrishnan, Subbaratnam

    2016-03-11

    Chitin is a major component of the exoskeleton and the peritrophic matrix of insects. It forms complex structures in association with different assortments of cuticle and peritrophic matrix proteins to yield biocomposites with a wide range of physicochemical and mechanical properties. The growth and development of insects are intimately coupled with the biosynthesis, turnover, and modification of chitin. The genes encoding numerous enzymes of chitin metabolism and proteins that associate with and organize chitin have been uncovered by bioinformatics analyses. Many of these proteins are encoded by sets of large gene families. There is specialization among members within each family, which function in particular tissues or developmental stages. Chitin-containing matrices are dynamically modified at every developmental stage and are under developmental and/or physiological control. A thorough understanding of the diverse processes associated with the assembly and turnover of these chitinous matrices offers many strategies to achieve selective pest control. PMID:26982439

  18. Chitin and Products of Its Hydrolysis in Vibrio cholerae Ecology.

    PubMed

    Markov, E Yu; Kulikalova, E S; Urbanovich, L Ya; Vishnyakov, V S; Balakhonov, S V

    2015-09-01

    The role of chitin and its hydrolysis products generated by Vibrio cholerae chitinases in mechanisms of its adaptation in water environments, metabolism, preservation, acquisition of pathogenic potential, and its epidemiological value are reviewed. Chitin utilization by V. cholerae as a source of energy, carbon, and nitrogen is described. Chitin association promotes biofilm formation on natural chitinous surfaces, increasing V. cholerae resistance to adverse factors in ecological niches: the human body and water environments with its inhabitants. Hydrolytic enzymes regulated by the corresponding genes result in complete chitin biodegradation by a chitinolytic catabolic cascade. Consequences of V. cholerae cell and chitin interaction at different hierarchical levels include metabolic and physiological cell reactions such as chemotaxis, cell division, biofilm formation, induction of genetic competence, and commensalic and symbiotic mutual relations with higher organisms, nutrient cycle, pathogenicity for humans, and water organisms that is an example of successful interrelation of bacteria and substratum in the ecology of the microorganism. PMID:26555464

  19. Chitin and Chitosan as Direct Compression Excipients in Pharmaceutical Applications

    PubMed Central

    Badwan, Adnan A.; Rashid, Iyad; Al Omari, Mahmoud M.H.; Darras, Fouad H.

    2015-01-01

    Despite the numerous uses of chitin and chitosan as new functional materials of high potential in various fields, they are still behind several directly compressible excipients already dominating pharmaceutical applications. There are, however, new attempts to exploit chitin and chitosan in co-processing techniques that provide a product with potential to act as a direct compression (DC) excipient. This review outlines the compression properties of chitin and chitosan in the context of DC pharmaceutical applications. PMID:25810109

  20. The battle for chitin recognition in plant-microbe interactions.

    PubMed

    Snchez-Vallet, Andrea; Mesters, Jeroen R; Thomma, Bart P H J

    2015-03-01

    Fungal cell walls play dynamic functions in interaction of fungi with their surroundings. In pathogenic fungi, the cell wall is the first structure to make physical contact with host cells. An important structural component of fungal cell walls is chitin, a well-known elicitor of immune responses in plants. Research into chitin perception has sparked since the chitin receptor from rice was cloned nearly a decade ago. Considering the widespread nature of chitin perception in plants, pathogens evidently evolved strategies to overcome detection, including alterations in the composition of cell walls, modification of their carbohydrate chains and secretion of effectors to provide cell wall protection or target host immune responses. Also non-pathogenic fungi contain chitin in their cell walls and are recipients of immune responses. Intriguingly, various mutualists employ chitin-derived signaling molecules to prepare their hosts for the mutualistic relationship. Research on the various types of interactions has revealed different molecular components that play crucial roles and, moreover, that various chitin-binding proteins contain dissimilar chitin-binding domains across species that differ in affinity and specificity. Considering the various strategies from microbes and hosts focused on chitin recognition, it is evident that this carbohydrate plays a central role in plant-fungus interactions. PMID:25725011

  1. Structure and function of enzymes acting on chitin and chitosan.

    PubMed

    Eijsink, Vincent; Hoell, Ingunn; Vaaje-Kolstada, Gustav

    2010-01-01

    Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4). PMID:21415904

  2. Isolation and characterization of chitin and chitosan from marine origin.

    PubMed

    Nwe, Nitar; Furuike, Tetsuya; Tamura, Hiroshi

    2014-01-01

    Nowadays, chitin and chitosan are produced from the shells of crabs and shrimps, and bone plate of squid in laboratory to industrial scale. Production of chitosan involved deproteinization, demineralization, and deacetylation. The characteristics of chitin and chitosan mainly depend on production processes and conditions. The characteristics of these biopolymers such as appearance of polymer, turbidity of polymer solution, degree of deacetylation, and molecular weight are of major importance on applications of these polymers. This chapter addresses the production processes and conditions to produce chitin, chitosan, and chito-oligosaccharide and methods for characterization of chitin, chitosan, and chito-oligosaccharide. PMID:25081074

  3. Advances in tetrahydropyrido[1,2-a]isoindolone (valmerins) series: Potent glycogen synthase kinase 3 and cyclin dependent kinase 5 inhibitors.

    PubMed

    Boulahjar, Rajâa; Ouach, Aziz; Bourg, Stéphane; Bonnet, Pascal; Lozach, Olivier; Meijer, Laurent; Guguen-Guillouzo, Christiane; Le Guevel, Rémy; Lazar, Saïd; Akssira, Mohamed; Troin, Yves; Guillaumet, Gérald; Routier, Sylvain

    2015-08-28

    An efficient synthetic strategy was developed to modulate the structure of the tetrahydropyridine isoindolone (Valmerin) skeleton. A library of more than 30 novel final structures was generated. Biological activities on CDK5 and GSK3 as well as cellular effects on cancer cell lines were measured for each novel compound. Additionally docking studies were performed to support medicinal chemistry efforts. A strong GSK3/CDK5 dual inhibitor (38, IC50 GSK3/CDK5 32/84 nM) was obtained. A set of highly selective GSK3 inhibitors was synthesized by fine-tuning structural modifications (29 IC50 GSK3/CDK5 32/320 nM). Antiproliferative effects on cells were correlated with the in vitro kinase activities and the best effects were obtained with lung and colon cell lines. PMID:26142492

  4. Rational design based on bioactive conformation analysis of pyrimidinylbenzoates as acetohydroxyacid synthase inhibitors by integrating molecular docking, CoMFA, CoMSIA, and DFT calculations.

    PubMed

    He, Yan-Zhen; Li, Yuan-Xiang; Zhu, Xiao-Lei; Xi, Zhen; Niu, Congwei; Wan, Jian; Zhang, Li; Yang, Guang-Fu

    2007-01-01

    Pyrimidinylthiobenzoates constitute an important kind of herbicides targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6), which catalyze the first common step in branched-chain amino acid biosynthesis. Due to the symmetry of 4,6-dimethoxypyrimidyl, there are two kinds of conformation of pyrimidinylthiobenzoates: one's phenyl is left-extending (named conformation-L); the other's phenyl is right-extending (named conformation-R). On the basis of the assumption that 3D quantitative structure-activity relationship (QSAR) models derived from the bioactive conformation should give the best result, a strategy of density-functional-theory-based 3D-QSAR was proposed to identify the bioactive conformation of pyrimidinylthiobenzoates by integrating the techniques of molecular docking, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and density functional theory calculation. The combination of three criteria of q2, r2, and r2pred obtained from CoMFA and CoMSIA analyses clearly indicated that conformation-R rather than conformation-L might be the bioactive conformation for pyrimidinylthiobenzoates. A further comparison between the two binding modes indicated that pyrimidinylthiobenzoates and sulfonylureas have very similar binding sites, such as Trp586, Arg380, and Pro192. However, Lys251 formed H bonds with sulfonylureas rather than pyrimidinylthiobenzoates. In addition, the orientation of phenyl groups of the two classes of compounds in the binding pocket were revealed to be opposite, which explained why the mutation of Pro192 displayed different sensitivity to sulfonylureas and pyrimidinylthiobenzoates. On the basis of the understanding of interactions between pyrimidinyl-thiobenzoates and AHAS, we designed and synthesized six 8-(4,6-dimethoxypyrimidin-2-yloxy)-4-methylphthalazin-1-one derivatives according to the 3D-QSAR models. The excellent correlation between the tested Ki values against wild-type A. thaliana acetohydroxyacid synthase and the predicted IC50 values demonstrated the high reliability of the established 3D-QSAR models. To our knowledge, this is the first report highlighting the binding mode of herbicidal pyrimidinylthiobenzoates, which consisted of the reported results of herbicide resistance. PMID:17887745

  5. Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.

    PubMed

    Verhoff, Moritz; Seitz, Stefanie; Paul, Michael; Noha, Stefan M; Jauch, Johann; Schuster, Daniela; Werz, Oliver

    2014-06-27

    The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3-30 ?M), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3?-acetoxy-8,24-dienetirucallic acid (6) and 3?-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 ?M, each. Structure-activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 ?M). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

  6. Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.

    PubMed Central

    Chang, A K; Duggleby, R G

    1998-01-01

    Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. PMID:9677339

  7. Tetra- and Pentacyclic Triterpene Acids from the Ancient Anti-inflammatory Remedy Frankincense as Inhibitors of Microsomal Prostaglandin E2 Synthase-1

    PubMed Central

    2014-01-01

    The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3–30 μM), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3α-acetoxy-8,24-dienetirucallic acid (6) and 3α-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 μM, each. Structure–activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 μM). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

  8. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    PubMed

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values. PMID:26586251

  9. Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzymes active site closure

    PubMed Central

    2012-01-01

    Background Human farnesyl pyrophosphate synthase (FPPS) controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzymes C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP). This conformational change, which occurs through a yet unclear mechanism, seals off the enzymes active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation. Results We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi), inorganic pyrophosphate (PPi), and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade. Conclusions In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the off position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores. PMID:23234314

  10. Activating the Wnt/?-Catenin Pathway for the Treatment of Melanoma Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3

    PubMed Central

    Atkinson, Jennifer M.; Rank, Kenneth B.; Zeng, Yi; Capen, Andrew; Yadav, Vipin; Manro, Jason R.; Engler, Thomas A.; Chedid, Marcio

    2015-01-01

    It has previously been observed that a loss of ?-catenin expression occurs with melanoma progression and that nuclear ?-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/?-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3? and GSK3? isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized ?-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that ?-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by ?-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored. PMID:25915038

  11. Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

    PubMed Central

    Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

  12. Timing of administration of dexamethasone or the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester, is critical for effective treatment of ischaemia-reperfusion injury to rat skeletal muscle.

    PubMed

    Zhang, B; Knight, K R; Dowsing, B; Guida, E; Phan, L H; Hickey, M J; Morrison, W A; Stewart, A G

    1997-08-01

    1. The effects of the nitric oxide synthase (NOS) inhibitors, NG-nitro-L-arginine-methyl ester (L-NAME), nitroiminoethyl-L-ornithine and S. methylisothiourea on skeletal muscle survival following 2 h of tourniquet ischaemia and 24 h of reperfusion were compared with those of the anti-inflammatory steroid, dexamethasone. 2. Administration of each of the NOS inhibitors or dexamethasone 30 min before reperfusion reduced the degree of skeletal muscle necrosis 24 h after reperfusion. 3. The influence of timing of drug administration was investigated. L-NAME administered 30 min before reperfusion, at 3 h after reperfusion, but not thereafter, significantly improved muscle survival compared with saline-treated controls. Dexamethasone administered 30 min before, or at 3 or 8 h after reperfusion, but not at 16 h, significantly improved muscle survival, but neither agent had protective effects when administered before ischaemia. 4. After 8 h of reperfusion of ischaemic skeletal muscle, cell-free homogenates contained Ca(2+)-independent (inducible) NOS activity which was reduced in dexamethasone-treated (2.5 mg/kg) rats. Furthermore, inducible NOS mRNA levels, as detected by reverse transcriptase-PCR, were increased after 8 h of reperfusion in saline, but not in dexamethasone-treated rats. 5. These data suggest a significant deleterious effect of endogenous NO which may be restricted to the first 3 h of the reperfusion phase of ischaemia-reperfusion injury, and raise the possibility of effective treatment of incipient reperfusion injury, even after several hours of reperfusion. PMID:9301432

  13. Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

    PubMed

    Semino, C E; Specht, C A; Raimondi, A; Robbins, P W

    1996-05-14

    The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis. PMID:8643441

  14. 4-Hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-one as inhibitors of tyrosyl-tRNA synthase: Synthesis, molecular docking and antibacterial evaluation

    NASA Astrophysics Data System (ADS)

    Sun, Juan; Liu, Jia-Jia; Zhou, Wei; Guo, Feng-Jiao; Wang, Xin-Yi; Zhu, Hai-Liang

    2014-01-01

    A series of novel 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors were synthesized. Of these compounds, 4-(naphthalen-1-ylmethyl)-5-oxo-2,5-dihydrothiophen-3-yl-2-(4-hydroxyphenyl)acetate (29) was the most potent. The binding model and structure-activity relationship indicate that replacement of phenyl acetate in the side chain of 29 with a substituent containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 29 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC50 value of 0.21 ?g/mL.

  15. Degradation and mineralization of chitin in an estuary

    SciTech Connect

    Boyer, J.

    1987-01-01

    A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described. /sup 14/C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D-(/sup 14/C)-glucosamine, allowing for its incorporation into the exoskeleton. Rates of chitin degradation and mineralization in estuarine water and sediments were determined as functions of temperature, inoculum source, and oxygen condition. Significant differences in rates between temperature treatments were evident. Q/sub 10/ values ranged from 1.2 to 2.5 for water and sediment, respectively. Increased incubation temperature also resulted in decreased lag times before onset of chitinoclastic bacterial growth and chitin degradation. The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other bacterial types. Actively growing chitinoclastic bacterial isolates produced primarily acetate, hydrogen, and carbon dioxide in broth culture.

  16. Granular chitin in the epidermis of nudibranch molluscs.

    PubMed

    Martin, Rainer; Hild, Sabine; Walther, Paul; Ploss, Kerstin; Boland, Wilhelm; Tomaschko, Karl-Heinz

    2007-12-01

    Chitin is usually found in stiff extracellular coatings typified by the arthropod exoskeleton, and is not associated with the soft, flexible mollusc skin. Here, we show, however, that chitin in nudibranch gastropods (Opisthobranchia, Mollusca) occurs as intracellular granules that fill the epidermal cells of the skin and the epithelial cells of the stomach. In response to nematocysts fired by tentacles of prey Cnidaria, the epidermal cells of eolid nudibranchs (Aeolidacea) release masses of chitin granules, which then form aggregates with the nematocyst tubules, having the effect of insulating the animal from the deleterious action of the Cnidaria tentacles. Granular chitin, while protecting the animal, does not interfere with the suppleness and flexibility of the skin, in contrast to the stiffness of chitin armor. The specialized epidermis enables nudibranchs to live with and feed on Cnidaria. PMID:18083970

  17. Chitin--the undisputed biomolecule of great potential.

    PubMed

    Tharanathan, Rudrapatnam N; Kittur, Farooqahmed S

    2003-01-01

    Of the truly abundant polysaccharides in Nature, only chitin has yet to find utilization in large quantity. Chitin is the second most abundant natural biopolymer derived from exoskeletons of crustaceans and also from cell walls of fungi and insects. Chitin is a linear beta 1,4-linked polymer of N-acetyl-D-glucosamine (GlcNAc), whereas chitosan, a copolymer of GlcNAc (approximately 20%) and glucosamine (GlcN, 80%) residues, is a product derived from de-N-acetylation of chitin in the presence of hot alkali. Chitosan is, in fact, a collective name representing a family of de-N-acetylated chitins deacetylated to different degrees. Both chitin/chitosan and their modified derivatives find extensive applications in medicine, agriculture, food, and non-food industries as well. They have emerged as a new class of physiological materials of highly sophisticated functions. Their application versatility is a great challenge to the scientific community and to industry. All these are the result of their versatile biological activity, excellent biocompatibility, and complete biodegradability in combination with low toxicity. Commercial availability of high-purity forms of chitin/chitosan and the continuous appearance of new types of chitin/chitosan derivatives with more and more useful and specific properties have led to an unlimited R&D efforts on this most versatile amino polysaccharide, chitin to find new applications, which are necessary to realize its full potential. Incidentally, this too has become an environmental priority. No doubt, chitin is surely an undisputed biomolecule of great potential. PMID:12587986

  18. Exoskeletal chitin scales isometrically with body size in terrestrial insects.

    PubMed

    Lease, Hilary M; Wolf, Blair O

    2010-06-01

    The skeletal system of animals provides the support for a variety of activities and functions. For animals such as mammals, which have endoskeletons, research has shown that skeletal investment (mass) scales with body mass to the 1.1 power. In this study, we ask how exoskeletal investment in insects scales with body mass. We measured the body mass and mass of exoskeletal chitin of 551 adult terrestrial insects of 245 species, with dry masses ranging from 0.0001 to 2.41 g (0.0002-6.13 g wet mass) to assess the allometry of exoskeletal investment. Our results showed that exoskeletal chitin mass scales isometrically with dry body mass across the Insecta as M(chitin) = a M(dry) (b), where b = 1.03 +/- 0.04, indicating that both large and small terrestrial insects allocate a similar fraction of their body mass to chitin. This isometric chitin-scaling relationship was also evident at the taxonomic level of order, for all insect orders except Coleoptera. We additionally found that the relative exoskeletal chitin investment, indexed by the coefficient, a, varies with insect life history and phylogeny. Exoskeletal chitin mass tends to be proportionally less and to increase at a lower rate with mass in flying than in nonflying insects (M(flying insect chitin) = -0.56 x M(dry) (0.97); M(nonflying insect chitin) = -0.55 x M(dry) (1.03)), and to vary with insect order. Isometric scaling (b = 1) of insect exoskeletal chitin suggests that the exoskeleton in insects scales differently than support structures of most other organisms, which have a positive allometry (b > 1) (e.g., vertebrate endoskeleton, tree secondary tissue). The isometric pattern that we document here additionally suggests that exoskeletal investment may not be the primary limit on insect body size. PMID:20235123

  19. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

    DOE PAGESBeta

    Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu; Nguyen, Cuong T.; Jedrzejczak, Robert P.; Joachimiak, Andrzej; Stacey, Gary

    2014-10-23

    Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5more » interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.« less

  20. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

    SciTech Connect

    Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu; Nguyen, Cuong T.; Jedrzejczak, Robert P.; Joachimiak, Andrzej; Stacey, Gary

    2014-10-23

    Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

  1. Effects of nitric oxide synthase inhibitor (L-NAME) on cytopathologic changes due to cholestasis in hepatic cells of adult male rats.

    PubMed

    Monsef, Alireza

    2012-12-01

    Obstructive cholestasis is associated with overproduction of endogenous opioids, nitric oxide (NO) and cytokines in the blood stream. Nitro-L-arginine methyl ester (L-NAME) administration decreases the NO serum level and it is able to reduce related complications. The aim of this research is to survey the effects of the NO inhibitor on complications relating to cholestasis in liver cells and intrahepatic biliary ducts. We used five groups of animals: control, sham-operated (surgical control), bile duct ligated (BDL) group, BDL and normal saline infused group, and BDL with L-NAME administrated group. After 3 weeks all animals were killed, histopathology of liver cells and intrahepatic biliary ducts were evaluated by hematoxylin-eosin (HE), PAS (periodic acid-Schiff) and trichrome staining. The status of inflammation and fibrosis was evaluated by the modified Knodell score system. Microscopic study of different groups showed that the necro-inflammatory score in the control group was 0.36, it was 1 in the sham-operated group and it raised to 15.2 in the cholestatic group. After administration of L-NAME it had a meaningful decrease to 7, but in the saline-treated group, the score was 16. L-NAME with the mentioned dose was capable of decreasing the serum nitric oxide level, although it is able to decrease the unfavorable complications of cholestatic jaundice. PMID:23359193

  2. Role of the anterior region of the third ventricle in the cardiovascular responses produced by systemic injection of a nitric oxide synthase inhibitor

    NASA Technical Reports Server (NTRS)

    Lewis, S. J.; Whalen, E. J.; Beltz, T. G.; Johnson, A. K.

    1999-01-01

    This study examined whether a prior electrolytic lesion of the tissue surrounding the anteroventral third ventricle (AV3V) would affect the increase in mean arterial blood pressure (MAP) and the fall in heart rate (HR) produced by systemic injection of the nitric oxide synthesis (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 25 micromol/kg, i.v.) in conscious rats. L-NAME produced a smaller increase in MAP in AV3V-lesion than in sham-lesion rats (+19+/-3 vs. +40+/-3 mmHg, respectively; P<0.05). In contrast, L-NAME produced similar falls in HR in the AV3V-lesion and sham-lesion rats (-103+/-15 vs. -97+/-8 bpm, respectively; P<0.05). These findings demonstrate that the L-NAME-induced pressor response is dependent upon the integrity of the AV3V region, whereas the L-NAME-induced bradycardia is not. Copyright 1999 Elsevier Science B. V.

  3. A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria.

    PubMed

    Rawls, Scott M; Rodriguez, Tonatiu; Baron, David A; Raffa, Robert B

    2006-07-12

    We previously reported that planarians (Dugesia dorotocephala) that have been exposed to cocaine for 1 h undergo abstinence-induced withdrawal when placed into cocaine-free, but not cocaine-containing, water. We now report that planarians also display dose-related abstinence-induced withdrawal following exposure to the synthetic cannabinoid agonist WIN 55212-2, but not its inactive enantiomer (WIN 55212-3). The withdrawal from WIN 55212-2 was manifested as a significant (P < 0.05) decrease in the rate of planarian spontaneous locomotor activity over a 5-min observation period, using a recently designed metric (pLMV). We also report that withdrawal from cocaine (80 microM) or WIN 55212-2 (10 microM) was attenuated by the selective inhibitor of nitric oxide synthesis L-NAME (L-nitro-arginine methyl ester), which had no effect of its own on pLMV. These results suggest a common NO-dependent pathway of withdrawal from cocaine and WIN 55212-2 in Planaria. PMID:16782070

  4. Structural alterations, pore generation, and deacetylation of ?- and ?-chitin submitted to steam explosion.

    PubMed

    Tan, Too Shen; Chin, Hui Yen; Tsai, Min-Lang; Liu, Chao-Lin

    2015-05-20

    The purpose of this study was to use an environmentally friendly steam explosion method to achieve ?- and ?-chitin structural alterations, pore generation, and deacetylation, enhancing the degree of deacetylation (DD) in chitin and extending its applications. The samples of ?- and ?-chitin possessing various moisture contents that were exploded at 9 kg/cm(2) exhibited higher DDs, lower densities, lower crystallinity and more porous structures compared to unexploded chitin. After explosion, ?-chitin exhibited a larger expansion ratio, lower crystallinity and contained a larger proportion of small-sized particles compared to ?-chitin. The highest DD values of exploded ?- and ?-chitin with 75% moisture content were 42.9% and 43.7%, respectively. The exploded chitin samples with lower moisture content exhibited lower DDs, densities, crystallinity indices, smaller particle sizes, and higher expansion ratios than the chitin samples with higher moisture content. The chitin samples with lower moisture content also contained larger and more numerous pores. PMID:25817675

  5. Timing and function of chitin synthesis in yeast.

    PubMed Central

    Cabib, E; Bowers, B

    1975-01-01

    A temperature-sensitive mutant of Saccharomyces cerevisiae, L-2-42, is blocked at 37 C at a stage of the cell cycle prior to septum formation. When single cells of the mutant are allowed to bud at 37 C in a medium containing tritiated glucose, a large incorporation of radioactivity into chitin takes place. Thus, the synthesis of chitin, the major component of the primary septum, is initiated in a phase of the cell cycle which precedes septum closure. This early period of chitin synthesis is not required for emergence and growth of buds because, in the wild type, budding takes place normally in the presence of concentrations of polyoxin D that effectively and specifically prevent chitin formation. However, at a later time a majority of these cells lyse, presumably because of the inability to form a septum. Polyoxin D also prevents the appearance of enhanced fluorescence at the junction between mother cell and bud, as observed in the presence of a brightener. Therefore, the fluorescence is due to chitin and its presence at the base of very early buds indicates that chitin synthesis begins at or shortly after bud emergence. A scheme for chitin synthesis and primary septum formation which embodies these and other results is presented. Images PMID:1104591

  6. Identification of 20-Hydroxyecdysone Late-Response Genes in the Chitin Biosynthesis Pathway

    PubMed Central

    Yao, Qiong; Zhang, Daowei; Tang, Bin; Chen, Jie; Chen, Jing; Lu, Liang; Zhang, Wenqing

    2010-01-01

    Background 20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis. Principal Findings Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes. Conclusions We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression. PMID:21124981

  7. A first-in-human phase I dose-escalation, pharmacokinetic, and pharmacodynamic evaluation of intravenous LY2090314, a glycogen synthase kinase 3 inhibitor, administered in combination with pemetrexed and carboplatin.

    PubMed

    Gray, Jhanelle E; Infante, Jeffrey R; Brail, Les H; Simon, George R; Cooksey, Jennifer F; Jones, Suzanne F; Farrington, Daphne L; Yeo, Adeline; Jackson, Kimberley A; Chow, Kay H; Zamek-Gliszczynski, Maciej J; Burris, Howard A

    2015-12-01

    Purpose LY2090314 (LY) is a glycogen synthase kinase 3 inhibitor with preclinical efficacy in xenograft models when combined with platinum regimens. A first-in-human phase 1 dose-escalation study evaluated the combination of LY with pemetrexed/carboplatin. Patients and Methods Forty-one patients with advanced solid tumors received single-dose LY monotherapy lead-in and 37 patients received LY (10-120mg) plus pemetrexed/carboplatin (500mg/m(2) and 5-6AUC, respectively) across 8 dose levels every 21days. Primary objective was maximum tolerated dose (MTD) determination; secondary endpoints included safety, antitumor activity, pharmacokinetics, and beta-catenin pharmacodynamics. Results MTD of LY with pemetrexed/carboplatin was 40mg. Eleven dose-limiting toxicities (DLTs) occurred in ten patients. DLTs during LY monotherapy occurred at ?40mg: grade 2 visual disturbance (n?=?1) and grade 3/4 peri-infusional thoracic pain during or shortly post infusion (n?=?4; chest, upper abdominal, and back pain). Ranitidine was added after de-escalation to 80mg LY to minimize peri-infusional thoracic pain. Following LY with pemetrexed/carboplatin therapy, DLTs included grade 3/4 thrombocytopenia (n?=?4) and grade 4 neutropenia (n?=?1). Best overall response by RECIST included 5 confirmed partial responses (non-small cell lung cancer [n?=?3], mesothelioma, and breast cancer) and 19 patients having stable disease. Systemic LY exposure was approximately linear over dose range studied. Transient upregulation of beta-catenin measured in peripheral blood mononuclear cells (PBMCs) occurred at 40mg LY. Conclusions The initial safety profile of LY2090314 was established. MTD LY dose with pemetrexed/carboplatin is 40mg IV every 3weeks plus ranitidine. Efficacy of LY plus pemetrexed/carboplatin requires confirmation in randomized trials. PMID:26403509

  8. Two squalene synthase inhibitors, E5700 and ER-119884, interfere with cellular proliferation and induce ultrastructural and lipid profile alterations in a Candida tropicalis strain resistant to fluconazole, itraconazole, and amphotericin B.

    PubMed

    Ishida, Kelly; Visbal, Gonzalo; Rodrigues, Juliany Cola Fernandes; Urbina, Julio A; de Souza, Wanderley; Rozental, Sonia

    2011-08-01

    Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16?g/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492nm, respectively, after 48h of incubation) of 1 and 4?g/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species. PMID:21264486

  9. Calcofluor white and Congo red inhibit chitin microfibril assembly of Poterioochromonas: evidence for a gap between polymerization and microfibril formation.

    PubMed

    Herth, W

    1980-11-01

    The influence of the light microscopical stains, Calcofluor white and Congo red, on the process of chitin microfibril formation of the chrysoflagellate alga Poterioochromonas stipitata was studied with light and electron microscopy. There is a concentration-dependent inhibition of lorica formation with both dyes. In the presence of the inhibitors malformed loricae are made, which do not show the usual ultrastructure and arrangement of the chitin microfibrils. Instead of long, laterally associated microfibrils, short rods or irregular networks of subelementary (15-25 A) fibrils are found. Microfibril assembly obviously takes place on the accessible outside of the plasma membrane. There must be a gap between the polymerization and microfibril formation reactions, allowing the stains to bind to the polymerized subunits. Thus, later association of these units to form microfibrils is disturbed. The microfibril-orienting mechanism also depends on normal microfibril formation. A model summarizing these hypotheses is suggested. PMID:7430250

  10. Calcofluor white and Congo red inhibit chitin microfibril assembly of Poterioochromonas: evidence for a gap between polymerization and microfibril formation

    PubMed Central

    1980-01-01

    The influence of the light microscopical stains, Calcofluor white and Congo red, on the process of chitin microfibril formation of the chrysoflagellate alga Poterioochromonas stipitata was studied with light and electron microscopy. There is a concentration-dependent inhibition of lorica formation with both dyes. In the presence of the inhibitors malformed loricae are made, which do not show the usual ultrastructure and arrangement of the chitin microfibrils. Instead of long, laterally associated microfibrils, short rods or irregular networks of subelementary (15-25 A) fibrils are found. Microfibril assembly obviously takes place on the accessible outside of the plasma membrane. There must be a gap between the polymerization and microfibril formation reactions, allowing the stains to bind to the polymerized subunits. Thus, later association of these units to form microfibrils is disturbed. The microfibril-orienting mechanism also depends on normal microfibril formation. A model summarizing these hypotheses is suggested. PMID:7430250

  11. [Purification and activity evaluation of methionine synthase].

    PubMed

    Guo, Ying; Li, Chao; Zhang, Zhi-Li; Tian, Chao; Wang, Xiao-Wei; Liu, Jun-Yi

    2012-11-01

    Methionine synthase (MS, EC2.1.1.13), a key enzyme in the folate metabolism area catalyzing methyl transfer from N5-methyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine, takes a core position in folate cycle, one-carbon-unit transfer and sculpture amino acid pathways. Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme was purified 609-fold to near homogeneity by batch chromatography on DE-52, anion-exchange chromatography on Q Sepharose Fast Flow and CHT-I hydroxyapatite column and was identified by SDS-PAGE and Western blotting. The enzyme activity was determined by spectrophotometric assay. In addition, the influencing factor and optimal reaction condition were performed. The steady state kinetic of rat liver methionine synthase was similar to that of other mammalian cobalamin-dependent methionine synthase which employed a Ping-Pong mechanism. The result indicated that cobalamin-dependent methionine synthase purified from rat liver is suitable for screening and studying methionine synthase specific inhibitors. PMID:23387078

  12. Emerging chitin and chitosan nanofibrous materials for biomedical applications

    NASA Astrophysics Data System (ADS)

    Ding, Fuyuan; Deng, Hongbing; Du, Yumin; Shi, Xiaowen; Wang, Qun

    2014-07-01

    Over the past several decades, we have witnessed significant progress in chitosan and chitin based nanostructured materials. The nanofibers from chitin and chitosan with appealing physical and biological features have attracted intense attention due to their excellent biological properties related to biodegradability, biocompatibility, antibacterial activity, low immunogenicity and wound healing capacity. Various methods, such as electrospinning, self-assembly, phase separation, mechanical treatment, printing, ultrasonication and chemical treatment were employed to prepare chitin and chitosan nanofibers. These nanofibrous materials have tremendous potential to be used as drug delivery systems, tissue engineering scaffolds, wound dressing materials, antimicrobial agents, and biosensors. This review article discusses the most recent progress in the preparation and application of chitin and chitosan based nanofibrous materials in biomedical fields.

  13. Chitin is a component of the Rhodnius prolixus midgut.

    PubMed

    Alvarenga, Evelyn S L; Mansur, Juliana F; Justi, Silvia A; Figueira-Mansur, Janaina; Dos Santos, Vivian M; Lopez, Sheila G; Masuda, Hatisaburo; Lara, Flavio A; Melo, Ana C A; Moreira, Monica F

    2016-02-01

    Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem. PMID:25910679

  14. Ab initio based conformational study of the crystalline ?-chitin.

    PubMed

    Petrov, Michal; Lymperakis, Liverios; Frik, Martin; Neugebauer, Jrg

    2013-01-01

    The equilibrium structure including the network of hydrogen bonds of an ?-chitin crystal is determined combining density-functional theory (DFT), self-consistent DFT-based tight-binding (SCC-DFTB), and empirical forcefield molecular dynamics (MD) simulations. Based on the equilibrium geometry several possible crystal conformations (local energy minima) have been identified and related to hydrogen bond patterns. Our results provide new insight and allow to resolve the contradicting ?-chitin structural models proposed by various experiments. PMID:23097227

  15. Chitin, Chitosan, and Its Derivatives for Wound Healing: Old and New Materials

    PubMed Central

    Azuma, Kazuo; Izumi, Ryotaro; Osaki, Tomohiro; Ifuku, Shinsuke; Morimoto, Minoru; Saimoto, Hiroyuki; Minami, Saburo; Okamoto, Yoshiharu

    2015-01-01

    Chitin (β-(1-4)-poly-N-acetyl-d-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. It is often converted to its more deacetylated derivative, chitosan. Previously, many reports have indicated the accelerating effects of chitin, chitosan, and its derivatives on wound healing. More recently, chemically modified or nano-fibrous chitin and chitosan have been developed, and their effects on wound healing have been evaluated. In this review, the studies on the wound-healing effects of chitin, chitosan, and its derivatives are summarized. Moreover, the development of adhesive-based chitin and chitosan are also described. The evidence indicates that chitin, chitosan, and its derivatives are beneficial for the wound healing process. More recently, it is also indicate that some nano-based materials from chitin and chitosan are beneficial than chitin and chitosan for wound healing. Clinical applications of nano-based chitin and chitosan are also expected. PMID:25780874

  16. Chitin, chitosan, and its derivatives for wound healing: old and new materials.

    PubMed

    Azuma, Kazuo; Izumi, Ryotaro; Osaki, Tomohiro; Ifuku, Shinsuke; Morimoto, Minoru; Saimoto, Hiroyuki; Minami, Saburo; Okamoto, Yoshiharu

    2015-01-01

    Chitin (?-(1-4)-poly-N-acetyl-D-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. It is often converted to its more deacetylated derivative, chitosan. Previously, many reports have indicated the accelerating effects of chitin, chitosan, and its derivatives on wound healing. More recently, chemically modified or nano-fibrous chitin and chitosan have been developed, and their effects on wound healing have been evaluated. In this review, the studies on the wound-healing effects of chitin, chitosan, and its derivatives are summarized. Moreover, the development of adhesive-based chitin and chitosan are also described. The evidence indicates that chitin, chitosan, and its derivatives are beneficial for the wound healing process. More recently, it is also indicate that some nano-based materials from chitin and chitosan are beneficial than chitin and chitosan for wound healing. Clinical applications of nano-based chitin and chitosan are also expected. PMID:25780874

  17. Comparison of Extraction Methods of Chitin from Ganoderma lucidum Mushroom Obtained in Submerged Culture

    PubMed Central

    Ospina lvarez, Sandra Patricia; Ramrez Cadavid, David Alexander; Ossa Orozco, Claudia Patricia; Zapata Ocampo, Paola; Atehorta, Luca

    2014-01-01

    The chitin was isolated from the Ganoderma lucidum submerged cultures mycelium as potential source of chitin under biotechnological processes. The extraction of chitin was carried out through 5 different assays which involved mainly three phases: pulverization of the mushroom, deproteinization of the mycelia with NaOH solution, and a process of decolorization with potassium permanganate and oxalic acid. The chitin contents extracted from 9-day mycelia were 413, 339, 87, 78, and 144?mg/g?1 (milligrams of chitin/grams of dry biomass) for A1, A2, A3, A4, and A5, respectively. Obtained chitin was characterized by X-Ray Diffraction (XRD), by Fourier transform infrared spectroscopy (FTIR), and by thermal analysis (TGA). The results showed that Ganoderma lucidum chitin has similar characteristic of chitin from different fonts. The advantage of the biotechnological processes and the fact that Ganoderma lucidum fungus may be used as a potential raw material for chitin production were demonstrated. PMID:24551839

  18. Processing of ?-chitin nanofibers by dynamic high pressure homogenization: characterization and antifungal activity against A. niger.

    PubMed

    Salaberria, Asier M; Fernandes, Susana C M; Diaz, Rene Herrera; Labidi, Jalel

    2015-02-13

    Chitin nano-objects become more interesting and attractive material than native chitin because of their usable form, low density, high surface area and promising mechanical properties. This work suggests a straightforward and environmentally friendly method for processing chitin nanofibers using dynamic high pressure homogenization. This technique proved to be a remarkably simple way to get ?-chitin into ?-chitin nanofibers from yellow lobster wastes with a uniform width (bellow 100 nm) and high aspect ratio; and may contributes to a major breakthrough in chitin applications. Moreover, the resulting ?-chitin nanofibers were characterized and compared with native ?-chitin in terms of chemical and crystal structure, thermal degradation and antifungal activity. The biological assays highlighted that the nano nature of chitin nanofibers plays an important role in the antifungal activity against Aspergillus niger. PMID:25458302

  19. Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation

    PubMed Central

    Ito, Ikuko; Yoneda, Toshikazu; Omura, Yoshihiko; Osaki, Tomohiro; Ifuku, Shinsuke; Saimoto, Hiroyuki; Azuma, Kazuo; Imagawa, Tomohiro; Tsuka, Takeshi; Murahata, Yusuke; Ito, Norihiko; Okamoto, Yoshiharu; Minami, Saburo

    2015-01-01

    Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm2), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation. PMID:26703629

  20. Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation.

    PubMed

    Ito, Ikuko; Yoneda, Toshikazu; Omura, Yoshihiko; Osaki, Tomohiro; Ifuku, Shinsuke; Saimoto, Hiroyuki; Azuma, Kazuo; Imagawa, Tomohiro; Tsuka, Takeshi; Murahata, Yusuke; Ito, Norihiko; Okamoto, Yoshiharu; Minami, Saburo

    2015-01-01

    Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation. PMID:26703629

  1. Digestibility of chitin in cod, Gadus morhua, in vivo

    NASA Astrophysics Data System (ADS)

    Danulat, Eva

    1987-12-01

    Sixteen cod, Gadus morhua (L.), were individually fed a single ration of shrimps, Crangon allmanni. Four fish were killed and examined 6, 12, 24 and 48 h after the fish had been fed. Chitinase activities were measured in the extracts of stomach contents, stomach tissue, pyloric caecae, intestinal contents and intestinal tissue. The level of enzyme activity in different parts of the digestive tract was shown to be dependent on the phase of the digestive process. High concentrations of the chitin degradation product N-acetyl-D-glucosamine were determined in the stomach and in the intestinal contents. Based on the chitin concentration in the food organisms and the individual food uptake, the amount of chitin consumed by each fish could be calculated. Only up to 9% of the ingested chitin was recovered from the intestinal contents of the fish at any given time after feeding (6, 12, 24 and 48 h). In addition, only 2.4% of the chitin consumed with the food could be recovered in the collected faeces of the fish. The 4 cod killed 48 h after feeding had completely emptied their stomach. Chitin digestion in these fish was calculated to have been 90%.

  2. Identification of Genes Putatively Involved in Chitin Metabolism and Insecticide Detoxification in the Rice Leaf Folder (Cnaphalocrocis medinalis) Larvae through Transcriptomic Analysis

    PubMed Central

    Yu, Hai-Zhong; Wen, De-Fu; Wang, Wan-Lin; Geng, Lei; Zhang, Yan; Xu, Jia-Ping

    2015-01-01

    The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest. PMID:26378520

  3. Identification of Genes Putatively Involved in Chitin Metabolism and Insecticide Detoxification in the Rice Leaf Folder (Cnaphalocrocis medinalis) Larvae through Transcriptomic Analysis.

    PubMed

    Yu, Hai-Zhong; Wen, De-Fu; Wang, Wan-Lin; Geng, Lei; Zhang, Yan; Xu, Jia-Ping

    2015-01-01

    The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest. PMID:26378520

  4. O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase

    PubMed Central

    Talukdar, Arindam; Zhao, Yujie; Lv, Wei; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Cushman, Mark

    2012-01-01

    Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction. PMID:22780198

  5. O-Nucleoside, S-nucleoside, and N-nucleoside probes of lumazine synthase and riboflavin synthase.

    PubMed

    Talukdar, Arindam; Zhao, Yujie; Lv, Wei; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Cushman, Mark

    2012-07-20

    Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction. PMID:22780198

  6. Structural differences between chitin and chitosan extracted from three different marine sources.

    PubMed

    Hajji, Sawssen; Younes, Islem; Ghorbel-Bellaaj, Olfa; Hajji, Rachid; Rinaudo, Marguerite; Nasri, Moncef; Jellouli, Kemel

    2014-04-01

    Three marine sources of chitin from Tunisia were investigated. Structural differences between α-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and β-chitin from cuttlefish (Sepia officinalis) bones were studied by the (13)C NMR, FTIR, and XRD diffractograms. The (13)C NMR analysis showed a splitting of the C3 and C5 carbon signals for α-chitin, while that of β-chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of α-chitin in the region of O-H, N-H and CO stretching regions. IR and (13)C NMR were used to determine the chitin degree of acetylation (DA). XRD analysis indicated that α-chitins were more crystalline polymorph than β-chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molecular masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source. PMID:24468048

  7. Anticancer effects of chitin and chitosan derivatives.

    PubMed

    Karagozlu, Mustafa Zafer; Kim, Se-Kwon

    2014-01-01

    Despite considerable progress in medical research, cancer is still one of the high-ranking causes of death in the world. It is the second most common cause of death due to disease after heart disease, and according to World Health Organization it will be the cause of death for more than 10 million people in 2020; therefore, one of the main research goals for researchers investigating new anticancer agents. But the major complication for the cancer cure without surgeries is side effects. Especially, cytotoxic anticancer chemotherapeutic agents generally produce severe side effects, while reducing host resistance to cancer and infections. Therefore, it is important to find new, powerful anticancer agents that are highly effective, biodegradable, and biocompatible. Chitin and chitosan are biopolymers which have unique structural possibilities for chemical and mechanical modifications to generate novel properties, functions. These biopolymers are biocompatible, biodegradable, and nontoxic, and their chemical properties allow them to be easily processed into gels, sponges, membranes, beads, and scaffolds forms also. Due to their unique properties, they are excellent candidates for cancer cure or cancer diagnosis. PMID:25081085

  8. ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila.

    PubMed

    Sobala, Lukasz F; Wang, Ying; Adler, Paul N

    2015-11-15

    Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects. PMID:26395478

  9. Development and binding mode assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-?-glutamyl-D-glutamic acid (BGC 945), a novel thymidylate synthase inhibitor that targets tumor cells.

    PubMed

    Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O'Connell, Joseph D; Griner, Sarah; Finer-Moore, Janet S; Stroud, Robert M

    2013-07-11

    N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-?-glutamyl-d-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with ?-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through ?-folate receptor (?-FR) transport. The ?-FR, a cell-surface receptor glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2'-deoxyuridine-5'-monophosphate, and a model for a similar complex with human TS. PMID:23710599

  10. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    PubMed Central

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-01-01

    Chitin-binding proteins (CBPs) are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP) from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs) of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton. PMID:26633500

  11. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin.

    PubMed

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-12-01

    Chitin-binding proteins (CBPs) are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP) from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs) of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton. PMID:26633500

  12. Microbial destruction of chitin in soils under different moisture conditions

    NASA Astrophysics Data System (ADS)

    Yaroslavtsev, A. M.; Manucharova, N. A.; Stepanov, A. L.; Zvyagintsev, D. G.; Sudnitsyn, I. I.

    2009-07-01

    The most favorable moisture conditions for the microbial destruction of chitin in soils are close to the total water capacity. The water content has the most pronounced effect on chitin destruction in soils in comparison with other studied substrates. It was found using gas-chromatographic and luminescent-microscopic methods that the maximum specific activity of the respiration of the chitinolytic community was at a rather low redox potential with the soil moisture close to the total water capacity. The range of moisture values under which the most intense microbial transformation of chitin occurred was wider in clayey and clay loamy soils as compared with sandy ones. The increase was observed due to the contribution of mycelial bacteria and actinomycetes in the chitinolytic complex as the soil moisture increased.

  13. Biosorption of gold from computer microprocessor leachate solutions using chitin.

    PubMed

    Crtes, Letcia N; Tanabe, Eduardo H; Bertuol, Daniel A; Dotto, Guilherme L

    2015-11-01

    The biosorption of gold from discarded computer microprocessor (DCM) leachate solutions was studied using chitin as a biosorbent. The DCM components were leached with thiourea solutions, and two procedures were tested for recovery of gold from the leachates: (1) biosorption and (2) precipitation followed by biosorption. For each procedure, the biosorption was evaluated considering kinetic, equilibrium, and thermodynamic aspects. The general order model was able to represent the kinetic behavior, and the equilibrium was well represented by the BET model. The maximum biosorption capacities were around 35 mg g(-1) for both procedures. The biosorption of gold on chitin was a spontaneous, favorable, and exothermic process. It was found that precipitation followed by biosorption resulted in the best gold recovery, because other species were removed from the leachate solution in the precipitation step. This method enabled about 80% of the gold to be recovered, using 20 g L(-1) of chitin at 298 K for 4 h. PMID:26188612

  14. Chitin-Induced Carbotype Conversion in Vibrio vulnificus?

    PubMed Central

    Neiman, Jana; Guo, Yunzhi; Rowe-Magnus, Dean A.

    2011-01-01

    As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. The most intensely studied of its virulence factors is the capsular polysaccharide (CPS). Over 100 CPS types have been identified, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second-most-abundant polysaccharide in nature, is known to induce competence in Vibrio species. Here, we show that the frequency of chitin-induced transformation in V. vulnificus varies by strain and that (GlcNAc)2 is the shortest chitin-derived polymer capable of inducing competence. Transformation frequencies (TFs) increased 8-fold when mixed-culture biofilms were exposed to a strain-specific lytic phage, suggesting that the lysis of dead cells during lytic infection increased the amount of extracellular DNA within the biofilm that was available for transfer. Furthermore, we show that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA (gDNA). The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. This suggested that the same mechanism governing the transfer of complete cps loci also contributed to their evolution by generating novel combinations of CPS biosynthesis genes. Since no evidence that cps loci were preferentially acquired during natural transformation (random transposon-tagged DNA was readily taken up in chitin transformation assays) exists, the phenomenon of chitin-induced transformation likely plays an important but general role in the evolution of this genetically promiscuous genus. PMID:21670169

  15. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins.

    PubMed Central

    Cabib, E

    1991-01-01

    Polyoxin D, nikkomycin X, and nikkomycin Z are all competitive inhibitors of chitin synthetase 2 (Chs2), the essential enzyme for primary septum formation in Saccharomyces cerevisiae, and of Chs1, a repair enzyme. However, Chs2 is more resistant to these antibiotics than Chs1. When Co2+, the best stimulator of Chs2, was used in the assay for this enzyme, the differences in the Ki values for nikkomycins between the two isozymes reached 3 orders of magnitude. These results point to differences in the active sites of the two isozymes. Polyoxin D was much more effective than nikkomycin Z in inhibiting cell growth. This underlines the importance of the choice of enzyme and of assay conditions when cell wall-synthesizing enzymes are used in screens for possible antifungal agents. PMID:2014972

  16. Glycan-functionalized fluorescent chitin nanocrystals for biorecognition applications.

    PubMed

    Zhou, Juan; Butchosa, Nria; Jayawardena, H Surangi N; Zhou, Qi; Yan, Mingdi; Ramstrm, Olof

    2014-04-16

    A new platform based on chitin nanocrystals has been developed for biorecognition applications. TEMPO-oxidized chitin nanocrystals (TCNs) were labeled with a fluorescent imidazoisoquinolinone dye, and simultaneously conjugated with carbohydrate ligands, resulting in dually functionalized TCNs. The biorecognition properties of the nanocrystals were probed with lectins and bacteria, resulting in selective interactions with their corresponding cognate carbohydrate-binding proteins, as visualized by optical, fluorescence, STEM, and TEM imaging. This represents a new approach to multifunctional nanomaterials based on naturally occurring polymers, holding high potential for biomedical applications. PMID:24625204

  17. Preparation of monolithic silica-chitin composite under extreme biomimetic conditions.

    PubMed

    Bazhenov, Vasilii V; Wysokowski, Marcin; Petrenko, Iaroslav; Stawski, Dawid; Sapozhnikov, Philipp; Born, Ren; Stelling, Allison L; Kaiser, Sabine; Jesionowski, Teofil

    2015-05-01

    Chitin is a widespread renewable biopolymer that is extensively distributed in the natural world. The high thermal stability of chitin provides an opportunity to develop novel inorganic-organic composites under hydrothermal synthesis conditions in vitro. For the first time, in this work we prepared monolithic silica-chitin composite under extreme biomimetic conditions (80C and pH 1.5) using three dimensional chitinous matrices isolated from the marine sponge Aplysina cauliformis. The resulting material was studied using light and fluorescence microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy. A mechanism for the silica-chitin interaction after exposure to these hydrothermal conditions is proposed and discussed. PMID:25701776

  18. Characterization of Chitin and Chitosan Molecular Structure in Aqueous Solution

    SciTech Connect

    Franca, Eduardo D.; Lins, Roberto D.; Freitas, Luiz C.; Straatsma, t. P.

    2008-11-08

    Molecular dynamics simulations have been used to characterize the structure of chitin and chitosan fibers in aqueous solutions. Chitin fibers, whether isolated or in the form of a β-chitin nanoparticle, adopt the so-called 2-fold helix with Φ and φ values similar to its crystalline state. In solution, the intramolecular hydrogen bond HO3(n)•••O5(n+1) responsible for the 2-fold helical motif is stabilized by hydrogen bonds with water molecules in a well-defined orientation. On the other hand, chitosan can adopt five distinct helical motifs and its conformational equilibrium is highly dependent on pH. The hydrogen bond pattern and solvation around the O3 atom of insoluble chitosan (basic pH) are nearly identical to these quantities in chitin. Our findings suggest that the solubility and conformation of these polysaccharides are related to the stability of the intrachain HO3(n)•••O5(n+1) hydrogen bond, which is affect by the water exchange around the O3-HO3 hydroxyl group.

  19. Dynamics of Gram-negative bacteria population density in a soil in the course of the succession initiated by chitin and cellulose

    NASA Astrophysics Data System (ADS)

    Konstantin, Ivanov; Lubov, Polyanskaya

    2014-05-01

    The functions of actinomycetes in polymer destruction in soil traditionally considered as the dominant, compare to another groups of bacteria. Gram-positive bacteria also have ecological functions in destruction of soil organic matter. The role of Gram-negative bacteria has been researched in the microbial succession in terms of polymers destruction, which are widely spreads in soils: chitin and cellulose. The method with nalidixic acid as an inhibitor of DNA division of Gram-negative bacteria was modified. By modified method microbial succession of Gram-negative bacteria in the different horizons of a chernozem under aerobic and anaerobic conditions was researched. Chitin and cellulose as the source of nutrients with moistening was used in experiments. The introduction of chitin had no positive effect on the population density of Gram-negative bacteria in a chernozem, but it advanced the date of their appearance in microbial succession: the maximum of Gram-negative bacteria population density was registered on the 3rd- 7th day of the experiment with adding chitin. Compare to the control, which one was without any nutrient adding this dynamics registered much earlier. Consequently, the introduction of chitin as an additional source of nutrition promoted revealing of the Gram-negative bacteria in soil already at the early stages of the succession. In the course of the succession, when the fungal mycelium begins to die off, the actinomycetic mycelium increases in length, i.e., Gram-negative bacteria are replaced at this stage with Gram-positive ones, the leading role among which belongs to actinomycetes. The growth rate of Gram-negative bacteria is higher than that of actinomycetes, so they start chitin utilization at the early stages of the succession, whereas actinomycetes dominate at the late stages. The population density of Gram-negative bacteria was lower under the anaerobic conditions as compared with that in the aerobic ones. The population density of Gram-negative bacteria in the lower layer of the A horizon of the chernozem and in the B horizon was slightly higher only in the case of the chitin introduction. When cellulose was introduced into the soil under aerobic conditions, the population density of Gram-negative bacteria in all the layers of the A horizon of the chernozem was maximal from the 14th to the 22nd day of the experiment. Simultaneously, an increase in the length of the actinomycetal mycelium was observed, as these organisms also perform cellulose hydrolysis in soils. The Gram-negative bacteria began to develop at the stage of the fungal mycelium destruction, which indirectly confirmed the chitinolytic activity of these bacteria.

  20. ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

    PubMed Central

    Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

    2015-01-01

    In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of ?/? subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the ? DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

  1. Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines.

    PubMed

    Holden, Whitney M; Fites, J Scott; Reinert, Laura K; Rollins-Smith, Louise A

    2014-01-01

    Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 μM. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis. PMID:24433676

  2. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    PubMed

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. PMID:26562543

  3. Biomimetic nanofibrous scaffolds: preparation and characterization of PGA/chitin blend nanofibers.

    PubMed

    Park, Ko Eun; Kang, Hyun Ki; Lee, Seung Jin; Min, Byung-Moo; Park, Won Ho

    2006-02-01

    Electrospinning of poly(glycolic acid) (PGA)/chitin blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated to fabricate biodegradable and biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun PGA/chitin blend nanofibers was investigated with a field emission scanning electron microscope. The PGA/chitin blend fibers have average diameters of around 140 nm, and their diameters have a distribution in the range 50-350 nm. The miscibility of PGA/chitin blend fibers was examined by differential scanning calorimetry. The PGA and chitin were immiscible in the as-spun nanofibrous structure. An in vitro degradation study of PGA/chitin blend nanofibers was conducted in phosphate-buffered saline, pH 7.2. It was found that the hydrolytic cleavage of PGA in the blend nanofibers was accelerated by the coexistence of hydrophilic chitin. To assay the cytocompatability and cell behavior on the PGA/chitin blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal fibroblasts seeded on the scaffolds were studied. Our results indicate that the PGA/chitin blend nanofibrous matrix, particularly the one that contained 25% PGA and 75% chitin with bovine serum albumin coating, could be a good candidate for tissue engineering scaffolds, because it has an excellent cell attachment and spreading for normal human fibroblasts. PMID:16471941

  4. Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

    PubMed

    Muramatsu, Masayoshi; Ohto, Chikara; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

    2008-09-01

    Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol. PMID:18636253

  5. Chiral nematic self-assembly of minimally surface damaged chitin nanofibrils and its load bearing functions.

    PubMed

    Oh, Dongyeop X; Cha, Yun Jeong; Nguyen, Hoang-Linh; Je, Hwa Heon; Jho, Yong Seok; Hwang, Dong Soo; Yoon, Dong Ki

    2016-01-01

    Chitin is one of the most abundant biomaterials in nature, with 10(10) tons produced annually as hierarchically organized nanofibril fillers to reinforce the exoskeletons of arthropods. This green and cheap biomaterial has attracted great attention due to its potential application to reinforce biomedical materials. Despite that, its practical use is limited since the extraction of chitin nanofibrils requires surface modification involving harsh chemical treatments, leading to difficulties in reproducing their natural prototypal hierarchical structure, i.e. chiral nematic phase. Here, we develop a chemical etching-free approach using calcium ions, called "natural way", to disintegrate the chitin nanofibrils while keeping the essential moiety for the self-assembly, ultimately resulting in the reproduction of chitin's natural chiral structure in a polymeric matrix. This chiral chitin nanostructure exceptionally toughens the composite. Our resultant chiral nematic phase of chitin materials can contribute to the understanding and use of the reinforcing strategy in nature. PMID:26988392

  6. Effect of Acetyl Group on Mechanical Properties of Chitin/Chitosan Nanocrystal: A Molecular Dynamics Study.

    PubMed

    Cui, Junhe; Yu, Zechuan; Lau, Denvid

    2016-01-01

    Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered. PMID:26742033

  7. Functionality of chitin as a direct compression excipient: an acetaminophen comparative study.

    PubMed

    Rojas, John; Ciro, Yhors; Correa, Luisa

    2014-03-15

    The particle and tableting properties of chitin extracted from shrimp exoskeletons were evaluated and compared with common excipients used for the preparation of tablets. Chitin offered more benefits in terms of functionality than calcium diphosphate, lactose monohydrate and pregelatinized starch. Further, highly plastic deforming materials such as sorbitol and PVP K30 and microcrystalline cellulose showed the best compactibility and dilution potential, whereas brittle deforming materials such as lactose monohydrate and calcium diphosphate were poorly compactable. Chitin had better compactibility than pregelatinized starch, calcium diphosphate and lactose monohydrate. Further, along with calcium diphosphate, chitin was the least sensitive material to compaction speed due to a combination of a plastic and brittle behavior. Moreover, chitin was less sensitive to magnesium stearate and possessed better acetaminophen loading capacity than pregelatinized starch, calcium diphosphate and lactose monohydrate. Chitin showed potential for use as a direct compression excipient. PMID:24528710

  8. Customizing Properties of ?-Chitin in Squid Pen (Gladius) by Chemical Treatments

    PubMed Central

    Ianiro, Alessandro; Di Giosia, Matteo; Fermani, Simona; Samor, Chiara; Barbalinardo, Marianna; Valle, Francesco; Pellegrini, Graziella; Biscarini, Fabio; Zerbetto, Francesco; Calvaresi, Matteo; Falini, Giuseppe

    2014-01-01

    The squid pen (gladius) from the Loligo vulgaris was used for preparation of ?-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The ?-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the ?-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the ?-chitin is modified, due to the intercalation of water molecules between ?-chitin sheets. Potential applications of these ?-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications. PMID:25517216

  9. Effect of soluble polysaccharides addition on rheological properties and microstructure of chitin nanocrystal aqueous dispersions.

    PubMed

    Tzoumaki, Maria V; Moschakis, Thomas; Biliaderis, Costas G

    2013-06-01

    Mixtures of chitin nanocrystal aqueous dispersions (at pH 3.0) with soluble polysaccharides of varying molecular features were examined rheologically and microscopically, under different conditions of biopolymer concentration, ionic strength, pH and temperature. The addition of non-adsorbing polysaccharides (guar gum, locust bean gum and xanthan) as well as oppositely charged (?-carrageenan) to a chitin nanocrystal dispersion, resulted in a network formation and the gel strength increased with the chitin nanocrystal concentration. In contrast, the chitin nanocrystal - chitosan or - pullulan mixed dispersions did not show any network formation (tan?>1) at the concentration range examined. An increase in ionic strength and pH also resulted in an enhanced elasticity of the chitin nanocrystal-guar gum dispersions. Furthermore, an increase in the elastic modulus, which was irreversible upon cooling, was observed upon heating the chitin nanocrystal-polysaccharide mixed dispersions. PMID:23618276

  10. Effect of addition of water-soluble chitin on amylose film.

    PubMed

    Suzuki, Shiho; Shimahashi, Katsumasa; Takahara, Junichi; Sunako, Michihiro; Takaha, Takeshi; Ogawa, Kozo; Kitamura, Shinichi

    2005-01-01

    Amylose films blended with chitosan, which were free from additives such as acid, salt, and plasticizer, were prepared by casting mixtures of an aqueous solution of an enzymatically synthesized amylose and that of water-soluble chitin (44.1% deacetylated). The presence of a small amount of chitin (less than 10%) increased significantly the permeability of gases (N2, O2, CO2, C2H4) and improved the mechanical parameters of amylose film; particularly, the elastic modulus and elongation of the blend films were larger than those of amylose or chitin films. No antibacterial activity was observed with either amylose or water-soluble chitin films. But amylose films having a small amount of chitin showed strong antibacterial action, suggesting a morphological change in water-soluble chitin on the film surface by blending with amylose molecule. These facts suggested the presence of a molecular complex of amylose and chitosan. PMID:16283751

  11. Surface morphology of chitin highly related with the isolated body part of butterfly (Argynnis pandora).

    PubMed

    Kaya, Murat; Bitim, Betl; Mujtaba, Muhammad; Koyuncu, Turgay

    2015-11-01

    This study was conducted to understand the differences in the physicochemical properties of chitin samples isolated from the wings and the other body parts except the wings (OBP) of a butterfly species (Argynnis pandora). The same isolation method was used for obtaining chitin specimens from both types of body parts. The chitin content of the wings (22%) was recorded as being much higher than the OBP (8%). The extracted chitin samples were characterized via FT-IR, TGA, XRD, SEM, and elemental analysis techniques. Results of these characterizations revealed that the chitins from both structures (wings and OBP) were very similar, except for their surface morphologies. SEM results demonstrated one type of surface morphology for the wings and four different surface morphologies for the OBP. Therefore, it can be hypothesized that the surface morphology of the chitin is highly related with the body part. PMID:26277749

  12. Effect of Acetyl Group on Mechanical Properties of Chitin/Chitosan Nanocrystal: A Molecular Dynamics Study

    PubMed Central

    Cui, Junhe; Yu, Zechuan; Lau, Denvid

    2016-01-01

    Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered. PMID:26742033

  13. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  14. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  15. Colloidal chitin nanogels: A plethora of applications under one shell.

    PubMed

    Vishnu Priya, M; Sabitha, M; Jayakumar, R

    2016-01-20

    Chitin nanogels (CNGs) are a relatively new class of natural polymeric nanomaterials which have a large potential in the field of drug delivery and nanotherapeutics. These nanogels being very biocompatible are non-toxic when internalized by cells. In this review various properties, preparation techniques and applications of CNGs have been described. CNGs because of their nano-size possess certain unique properties which enable them to be used in a number of biomedical applications. CNGs are prepared by simple regeneration technique without using any cross-linkers. Various polymers, drugs and fluorescent dyes can be blended or incorporated or labelled with the chitin hydrogel network. Drugs and molecules encapsulated within CNGs can be used for targeted delivery, in vivo monitoring or even for therapeutic purposes. Here various applications of CNGs in the field of drug delivery, imaging, sensing and therapeutics have been discussed. PMID:26572393

  16. Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

    PubMed Central

    Weigel, Paul H.

    2015-01-01

    Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers. PMID:26472958

  17. Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae Tü901 That Interferes with Growth Polarity

    PubMed Central

    Bormann, Christiane; Baier, Daniel; Hörr, Ingmar; Raps, Claudia; Berger, Jürgen; Jung, Günther; Schwarz, Heinz

    1999-01-01

    The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against Paecilomyces variotii in agar diffusion assays. The 384-bp afp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for Streptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of Aspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching. PMID:10601197

  18. Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

    PubMed

    Thompson, Keith; Dunford, James E; Ebetino, Frank H; Rogers, Michael J

    2002-01-18

    We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase. PMID:11785983

  19. Recent trends in biological extraction of chitin from marine shell wastes: a review.

    PubMed

    Kaur, Surinder; Dhillon, Gurpreet Singh

    2015-03-01

    The natural biopolymer chitin and its deacetylated product chitosan are widely used in innumerable applications ranging from biomedicine, pharmaceuticals, food, agriculture and personal care products to environmental sector. The abundant and renewable marine processing wastes are commercially exploited for the extraction of chitin. However, the traditional chitin extraction processes employ harsh chemicals at elevated temperatures for a prolonged time which can harm its physico-chemical properties and are also held responsible for the deterioration of environmental health. In view of this, green extraction methods are increasingly gaining popularity due to their environmentally friendly nature. The bioextraction of chitin from crustacean shell wastes has been increasingly researched at the laboratory scale. However, the bioextraction of chitin is not currently exploited to its maximum potential on the commercial level. Bioextraction of chitin is emerging as a green, cleaner, eco-friendly and economical process. Specifically in the chitin extraction, microorganisms-mediated fermentation processes are highly desirable due to easy handling, simplicity, rapidity, controllability through optimization of process parameters, ambient temperature and negligible solvent consumption, thus reducing environmental impact and costs. Although, chitin production from crustacean shell waste through biological means is still at its early stage of development, it is undergoing rapid progress in recent years and showing a promising prospect. Driven by reduced energy, wastewater or solvent, advances in biological extraction of chitin along with valuable by-products will have high economic and environmental impact. PMID:24083454

  20. Nanostructured biocomposite films of high toughness based on native chitin nanofibers and chitosan

    NASA Astrophysics Data System (ADS)

    Mushi, Ngesa; Utsel, Simon; Berglund, Lars

    2014-11-01

    Chitosan is widely used in films for packaging applications. Chitosan reinforcement by stiff particles or fibers is usually obtained at the expense of lowered ductility and toughness. Here, chitosan film reinforcement by a new type of native chitin nanofibers is reported. Films are prepared by casting from colloidal suspensions of chitin in dissolved chitosan. The nanocomposite films are chitin nanofiber networks in chitosan matrix. Characterization is carried out by dynamic light scattering, quartz crystal microbalance, field emission scanning electron microscopy, tensile tests and dynamic mechanical analysis. The nanostructured biocomposite was produced in volume fractions of 0, 8, 22 and 56% chitin nanofibers. Favorable chitin-chitosan synergy for colloidal dispersion is demonstrated. Also, lowered moisture sorption is observed for the composites, probably due to the favorable chitin-chitosan interface. The highest toughness (area under stress-strain curve) was observed at 8 vol% chitin content. The toughening mechanisms and the need for well-dispersed chitin nanofibers is discussed. Finally, desired structural characteristics of ductile chitin biocomposites are discussed.

  1. Discussion remarks on the role of wood and chitin constituents during carbonization

    NASA Astrophysics Data System (ADS)

    Ilnicka, Anna; Lukaszewicz, Jerzy

    2015-03-01

    Nature is a source of some biomaterials like wood and chitin which can be successfully transformed into chars of advanced structural/surface parameters. The manuscript is discursive and suggests that particular components of the materials (cellulose, lignin, hemicellulose, alfa-chitin fibrils, mineral-protein matrix) play a specific role in the manufacturing of porous chars. It is proposed that some of the components (hemicellulose and mineral-protein matrixes) behave like a natural soft template during carbonization of wood and chitin. It is suggested why particular components and derivatives of wood and chitin (cellulose and chitosan) can not form porous carbonaceous matrixes when are carbonized separately.

  2. A comparative study of sorption of chromium (III) onto chitin and chitosan

    NASA Astrophysics Data System (ADS)

    Singh, Pooja; Nagendran, R.

    2014-07-01

    Heavy metals have always been the most hazardous components in the wastewater of industries like electroplating, automobiles, mining facilities and fertilizer manufacturers. Treatment of heavy metal laden wastewater requires expensive operational and maintenance systems. Food processing industries create a huge amount of shell waste which is sold to poultry farms in powdered form but the quantity thus used is still not comparable to the left over waste. The shell contains chitin which acts as an adsorbent for the heavy metals and can be used to treat heavy metal wastewater. The paper presents a study on the use of chitin and its processed product, chitosan, to remove chromium. Shake flask experiment was conducted to compare the adsorptive capacity of chitin and chitosan for chromium removal from simulated solution and isotherm studies were carried out. The studies showed that the chitosan was a better adsorbent than chitin. Both chitin and chitosan gave best adsorption results at pH 3. Chitin exhibited maximum chromium removal of 49.98 % in 20 min, whereas chitosan showed 50 % removal efficiency at a contact time of 20 min showing higher adsorptive capacity for chromium than chitin. The Langmiur and Freundlich isotherm studies showed very good adsorption capacity and monolayer interaction according to the regression coefficient 0.973 for chitosan and 0.915 for chitin. The regression coefficient for Freundlich isotherm was 0.894 and 0.831 for chitosan and chitin, respectively.

  3. Nanostructured biocomposite films of high toughness based on native chitin nanofibers and chitosan

    PubMed Central

    Mushi, Ngesa E.; Utsel, Simon; Berglund, Lars A.

    2014-01-01

    Chitosan is widely used in films for packaging applications. Chitosan reinforcement by stiff particles or fibers is usually obtained at the expense of lowered ductility and toughness. Here, chitosan film reinforcement by a new type of native chitin nanofibers is reported. Films are prepared by casting from colloidal suspensions of chitin in dissolved chitosan. The nanocomposite films are chitin nanofiber networks in chitosan matrix. Characterization is carried out by dynamic light scattering, quartz crystal microbalance, field emission scanning electron microscopy, tensile tests and dynamic mechanical analysis. The polymer matrix nanocomposites were produced in volume fractions of 8, 22, and 56% chitin nanofibers. Favorable chitin-chitosan synergy for colloidal dispersion is demonstrated. Also, lowered moisture sorption is observed for the composites, probably due to the favorable chitin-chitosan interface. The highest toughness (area under stress-strain curve) was observed at 8 vol% chitin content. The toughening mechanisms and the need for well-dispersed chitin nanofibers is discussed. Finally, desired structural characteristics of ductile chitin biocomposites are discussed. PMID:25478558

  4. Elevated Chitin Content Reduces the Susceptibility of Candida Species to Caspofungin

    PubMed Central

    Walker, Louise A.; Gow, Neil A. R.

    2013-01-01

    The echinocandin antifungal drugs inhibit synthesis of the major fungal cell wall polysaccharide β(1,3)-glucan. Echinocandins have good efficacy against Candida albicans but reduced activity against other Candida species, in particular Candida parapsilosis and Candida guilliermondii. Treatment of Candida albicans with a sub-MIC level of caspofungin has been reported to cause a compensatory increase in chitin content and to select for sporadic echinocandin-resistant FKS1 point mutants that also have elevated cell wall chitin. Here we show that elevated chitin in response to caspofungin is a common response in various Candida species. Activation of chitin synthesis was observed in isolates of C. albicans, Candida tropicalis, C. parapsilosis, and C. guilliermondii and in some isolates of Candida krusei in response to caspofungin treatment. However, Candida glabrata isolates demonstrated no exposure-induced change in chitin content. Furthermore, isolates of C. albicans, C. krusei, C. parapsilosis, and C. guilliermondii which were stimulated to have higher chitin levels via activation of the calcineurin and protein kinase C (PKC) signaling pathways had reduced susceptibility to caspofungin. Isolates containing point mutations in the FKS1 gene generally had higher chitin levels and did not demonstrate a further compensatory increase in chitin content in response to caspofungin treatment. These results highlight the potential of increased chitin synthesis as a potential mechanism of tolerance to caspofungin for the major pathogenic Candida species. PMID:23089748

  5. Natural porous and nano fiber chitin structure from Gammarus argaeus (Gammaridae Crustacea)

    PubMed Central

    Kaya, Murat; Tozak, Kabil Özcan; Baran, Talat; Sezen, Göksal; Sargin, Idris

    2013-01-01

    Chitin and its derivatives are commercially important biopolymers due to their applications in medicine, agriculture, water treatment, cosmetics and various biotechnological areas. Since chitin and its derivatives exhibit different chemical and physical properties depending on the source and isolation method, there is a growing demand for new chitin sources other than crab and shrimp worldwide. In this study Gammarus, a Crustacea, was investigated as a novel chitin source. Gammarus, which belongs to the family Gammaridae Crustacea, lives in the bottom of aquatic ecosystems. More than 200 species are known worldwide. One of these species, G. argaeus was investigated for chitin isolation. The alpha chitin isolated from G. argaeus was characterized by using analysis techniques such as infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). All these analyses confirmed that the isolated chitin from G. argaeus was in the alpha form. Furthermore, we described that dry weight of this species contained 11-12 % chitin. SEM examination of the isolated α-chitin revealed that it was composed of nanofibrils (15-55 nm) and pores (about 150 nm). PMID:26966425

  6. Dimers of mitochondrial ATP synthase form the permeability transition pore.

    PubMed

    Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D; Petronilli, Valeria; Zoratti, Mario; Szab, Ildik; Lippe, Giovanna; Bernardi, Paolo

    2013-04-01

    Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

  7. Synthesis and antibacterial activity of cinnamaldehyde acylhydrazone with a 1,4-benzodioxan fragment as a novel class of potent ?-ketoacyl-acyl carrier protein synthase III (FabH) inhibitor.

    PubMed

    Song, Xiaoda; Yang, Yushun; Zhao, Jing; Chen, Yangjian

    2014-01-01

    Fatty acid biosynthesis is essential for bacterial survival. ?-Ketoacyl-acyl carrier protein (ACP) synthase III (FabH), is a particularly attractive antibacterial target, since it is central to the initiation of fatty acid biosynthesis. Three series of 21 cinnamaldehyde acylhydrazone derivatives, A3-9, B3-9, and C3-9, were synthesized and evaluated for FabH-inhibitory activity. Compound B6 showed the most potent biological activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis (minimum inhibitory concentrations (MICs) values: 1.56-3.13 g/mL) and was comparable with the positive control. Docking simulation by positioning compound B6 in the FabH structure active site was performed to explore the possible binding model. PMID:25196128

  8. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  9. Chitin: Formation of Helically Structured Chitin/CaCO3 Hybrids through an Approach Inspired by the Biomineralization Processes of Crustacean Cuticles (Small 38/2015).

    PubMed

    Matsumura, Shunichi; Kajiyama, Satoshi; Nishimura, Tatsuya; Kato, Takashi

    2015-10-01

    Biomineral-inspired hybrids forming helically ordered structures are developed by T. Kato and co-workers on page 5127. These helical hybrids consist of liquid-crystalline chitin and CaCO3 . They resemble the structures of crustacean cuticles such as the exoskeleton of a lobster or crayfish. These hybrids are formed through CaCO3 crystallization on the liquidcrystalline chitin templates. Polymer-stabilized amorphous CaCO3 is incorporated into the liquid-crystalline chitin templates. This approach is useful for the development of new hierarchical hybrid materials from abundant natural resources. PMID:26450162

  10. SEM characterization of anatomical variation in chitin organization in insect and arthropod cuticles.

    PubMed

    Chandran, Rakkiyappan; Williams, Lee; Hung, Albert; Nowlin, Kyle; LaJeunesse, Dennis

    2016-03-01

    The cuticles of insects and arthropods have some of the most diverse material properties observed in nature, so much so that it is difficult to imagine that all cutciles are primarily composed of the same two materials: a fibrous chitin network and a matrix composed of cuticle proteins. Various factors contribute to the mechanical and optical properties of an insect or arthropod cuticle including the thickness and composition. In this paper, we also identified another factor that may contribute to the optical, surface, and mechanical properties of a cuticle, i.e. the organization of chitin nanofibers and chitin fiber bundles. Self-assembled chitin nanofibers serve as the foundation for all higher order chitin structures in the cuticles of insects and other arthropods via interactions with structural cuticle proteins. Using a technique that enables the characterization of chitin organization in the cuticle of intact insects and arthropod exoskeletons, we demonstrate a structure/function correlation of chitin organization with larger scale anatomical structures. The chitin scaffolds in cuticles display an extraordinarily diverse set of morphologies that may reflect specific mechanical or physical properties. After removal of the proteinaceous and mineral matrix of a cuticle, we observe using SEM diverse nanoscale and micro scale organization of in-situ chitin in the wing, head, eye, leg, and dorsal and ventral thoracic regions of the periodical cicada Magicicada septendecim and in other insects and arthropods. The organization of chitin also appears to have a significant role in the organization of nanoscale surface structures. While microscale bristles and hairs have long been known to be chitin based materials formed as cellular extensions, we have found a nanostructured layer of chitin in the cuticle of the wing of the dog day annual cicada Tibicen tibicens, which may be the scaffold for the nanocone arrays found on the wing. We also use this process to examine the chitin organizations in the fruit fly, Drosophila melanogaster, and the Atlantic brown shrimp, Farfantepenaeus aztecus. Interestingly many of the homologous anatomical structures from diverse arthropods exhibit similar patterns of chitin organization suggesting that a common set of parameters, govern chitin organization. PMID:26774746

  11. Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo

    PubMed Central

    Lee, Keunsook K.; MacCallum, Donna M.; Jacobsen, Mette D.; Walker, Louise A.; Odds, Frank C.

    2012-01-01

    Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations. PMID:21986821

  12. Ceramide synthases in biomedical research.

    PubMed

    Cingolani, Francesca; Futerman, Anthony H; Casas, Josefina

    2016-05-01

    Sphingolipid metabolism consists of multiple metabolic pathways that converge upon ceramide, one of the key molecules among sphingolipids (SLs). In mammals, ceramide synthesis occurs via N-acylation of sphingoid backbones, dihydrosphingosine (dhSo) or sphingosine (So). The reaction is catalyzed by ceramide synthases (CerS), a family of enzymes with six different isoforms, with each one showing specificity towards a restricted group of acyl-CoAs, thus producing ceramides (Cer) and dihydroceramides (dhCer) with different fatty acid chain lengths. A large body of evidence documents the role of both So and dhSo as bioactive molecules, as well as the involvement of dhCer and Cer in physiological and pathological processes. In particular, the fatty acid composition of Cer has different effects in cell biology and in the onset and progression of different diseases. Therefore, modulation of CerS activity represents an attractive target in biomedical research and in finding new treatment modalities. In this review, we discuss functional, structural and biochemical features of CerS and examine CerS inhibitors that are currently available. PMID:26248326

  13. Mycelial development preceding basidioma formation in Moniliophthora perniciosa is associated to chitin, sugar and nutrient metabolism alterations involving autophagy.

    PubMed

    Gomes, Dayane Santos; Lopes, Maíza Alves; Menezes, Sara Pereira; Ribeiro, Lidiane Figueredo; Dias, Cristiano Villela; Andrade, Bruno Silva; de Jesus, Raildo Mota; Pires, Acassia Benjamin Leal; Goes-Neto, Aristóteles; Micheli, Fabienne

    2016-01-01

    We identified and characterized two chitinases, named MpCHIT1 and MpCHIT2, from the fungus Moniliophthora perniciosa - the etiologic agent of witches' broom disease in cacao tree (Theobroma cacao L.) - during its development, mainly in the mycelia phases preceding the basidioma formation. The expression of MpCHIT1 and MpCHIT2, together with MpCHS and MpATG8 (chitin synthase and autophagy genes, respectively), was analyzed during the M. perniciosa growth and development on bran-based solid medium as well as in liquid medium containing H2O2 or rapamycin (oxidative and nutritional related-autophagy stress agents, respectively). In order to link the expression of chitin metabolism-related genes to nutritional composition influencing fungus development, we also quantified total and reduced sugars, as well as macro- and micronutrients in the bran-based solid medium. The expression analysis showed that the MpCHS expression increased through mycelial development and then decreased in the primordium and basidioma phases, while the expression of MpCHIT1 and MpCHIT2 was higher in basidioma and primordium phases, respectively. Moreover, the expression pattern of MpCHIT1 and MpCHIT2 is distinct, the second correlated with the MpATG8 expression pattern and possibly with autophagy process, while the first may be related to the basidioma formation. The quantification of total and reduced sugars, as well as macro- and micronutrients supported the idea that the cell wall restructuration due to MpCHS, MpCHIT1 and MpCHIT2 is related to stress and fungal nutrient reallocation, allowing the formation and development of the basidioma. Experiments involving M. perniciosa growth on liquid medium containing H2O2 or rapamycin showed that MpCHIT1 and MpCHIT2 were over-expressed in response to oxidative but also to nutritional related-autophagy stresses. Interestingly, the expression level of MpCHS, MpCHIT1 and MpCHIT2 in presence of rapamycin is similar to the one observed in the primordium and basidioma from bran-based solid medium. The analysis of the overall data allowed designing a general scheme of chitin metabolism and autophagy during M. perniciosa development, focusing on the mycelium phases as crucial and environmentally influenced steps preceding the primordium and basidioma formation. These data support the idea that the nutritional environment of M. perniciosa influences its development and life cycle. PMID:26701307

  14. Recent development of two chitinase inhibitors, Argifin and Argadin, produced by soil microorganisms

    PubMed Central

    Hirose, Tomoyasu; Sunazuka, Toshiaki; Ōmura, Satoshi

    2010-01-01

    Chitin, the second most abundant polysaccharide in nature, occurs in fungi, some algae and many invertebrates, including insects. Thus, chitin synthesis and degradation could represent specific targets for fungicides and insecticides. Chitinases hydrolyze chitin into oligomers of N-acetyl-d-glucosamine at key points in the life cycles of organisms, consequently, chitinase inhibitors have become subject of increasing interest. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products, the synthesis of lead compounds via computer-aided rational molecular design, and discovery methods that generate only highly-active compounds using a kinetic target(chitinase)-guided synthesis approach (termed in situ click chemistry) are described. PMID:20154467

  15. Distribution of naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana: molecular events in prey capture.

    PubMed

    Raj, Gopan; Kurup, Rajani; Hussain, Abdul Azeez; Baby, Sabulal

    2011-11-01

    Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana. PMID:21862483

  16. Enantioseparation characteristics of tadalafil and its intermediate on chitin derived chiral stationary phases.

    PubMed

    Zhang, Juan; Wang, Zhao-Qun; Wang, Xiao-Chen; Zhang, Jun-Jun; Bai, Zheng-Wu; Chen, Wei

    2015-08-21

    Due to the low solubility and swelling properties of chitin bis(arylcarbamate) in most organic solvents, the chiral stationary phases (CSPs) prepared from chitin derivatives can be analyzed with a wide range of solvents. In order to develop new CSPs of chitin derivatives with halogen groups, chitin was derivatized with three different phenyl isocyanates to obtain chitin bis(4-trifluoromethoxyphenylcarbamate), chitin bis(3-chloro-4-methylphenylcarbamate) and chitin bis(4-chloro-3-trifluoromethylphenylcarbamate). Then, the three chitin derivatives were coated on macroporous 3-aminopropyl-functionalized silica gel to obtain three CSPs 1-3 respectively. These CSPs showed good enantioseparation capabilities towards tadalafil and its intermediate. Therefore, these CSPs are potentially applied for the enantioseparation and determination of tadalafil and its intermediate. It was also found that the retention times of some enantiomers were prolonged in the presence of their diastereoisomers or structural analogues and as a result, the resolution was improved to some extent. Hence, in the enantioseparation of a compound with two chiral centers, the resolution is hopefully improved by adding a diastereoisomer or a structural analogue. PMID:26115207

  17. Effect of chitin on the antagonistic activity of Cryptococcus laurentii against Penicillium expansum in pear fruit.

    PubMed

    Yu, Ting; Wang, Lianping; Yin, Yun; Wang, Yixi; Zheng, Xiaodong

    2008-02-29

    This study was designed to evaluate the impact of chitin on the antagonistic activity of Cryptococcus laurentii against the postharvest blue mold rot caused by Penicillium expansum in pear fruit. The results showed that the antagonistic activity of C. laurentii obtained from the culture media of nutrient yeast dextrose broth (NYDB) amended with chitin at 0.5-1.0% was improved greatly compared with the case that without chitin. The addition of chitin to NYDB did not influence the growth of C. laurentii, however, its population was found to increase rapidly thereafter in pear fruit wounds compared to that harvested from NYDB without chitin. Moreover, the cell-free filtrate of the chitin-supplement culture media in which the yeast was incubated for 24 h emerged a direct antifungal activity against P. expansum in pear fruit wounds, with the associated high level of chitinase activity. These results suggested that the use of chitin may be an effective method to induce the antagonistic activity of C. laurentii. To our knowledge, this is the first report regarding the chitin could enhance the efficacy of postharvest biocontrol yeasts. PMID:18162192

  18. Hydrophobic modification on surface of chitin sponges for highly effective separation of oil.

    PubMed

    Duan, Bo; Gao, Huimin; He, Meng; Zhang, Lina

    2014-11-26

    A highly hydrophobic and oleophilic chitin sponge was synthesized, for the first time, via a freeze-dried method and then by using a thermal chemical vapor deposition of methyltrichlorosilane (MTCS) at different relative humidity. Fourier-transform infrared, energy-dispersive X-ray spectra, and scanning electron microscopy confirmed that the silanization occurred on the pore wall surface of the chitin sponge. The MTCS-coated chitin sponge had interconnected open-cell structures with the average pore size from 20 to 50 ?m, and the MTCS nanofilaments immobilized on the chitin matrix, leading to the high hydrophobicity, as a result of the existence of a solid/air composite rough surface. Cyclic compression test indicated that the hydrophobic chitin sponges exhib