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Sample records for chitin synthase inhibitor

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

    PubMed Central

    Schönitzer, 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

  2. 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

  3. Design, synthesis and evaluation of novel quinazoline-2,4-dione derivatives as chitin synthase inhibitors and antifungal agents.

    PubMed

    Ji, Qinggang; Yang, Dan; Wang, Xin; Chen, Chunyan; Deng, Qiao; Ge, Zhiqiang; Yuan, Lvjiang; Yang, Xiaolan; Liao, Fei

    2014-07-01

    A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized, and characterized by (1)H NMR, (13)C NMR and MS spectral data. Their inhibition against chitin synthase (CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j, 5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency among these compounds, had a half-inhibition concentration (IC50) of 0.08mmol/L, while polyoxin B as positive drug had IC50 of 0.18mmol/L. These IC50 values of compounds 5i, 5m, 5n, and 5s were greater than 0.75mmol/L, which revealed that those compounds had weak inhibition activity against CHS. Moreover, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the minimum-inhibition-concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to their IC50 values against CHS. Furthermore, these compounds had negligible actions to bacteria. Therefore, these compounds were promising selective antifungal agents. PMID:24856180

  4. 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; Grbi?, 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

  5. 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...

  6. 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...

  7. 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.

  8. 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, ...

  9. Early Divergence, Broad Distribution, and High Diversity of Animal Chitin Synthases

    PubMed Central

    Zakrzewski, Anne-C.; Weigert, Anne; Helm, Conrad; Adamski, Marcin; Adamska, Maja; Bleidorn, Christoph; Raible, Florian; Hausen, Harald

    2014-01-01

    Even though chitin is one of the most abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding new light on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. The most extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs in molluscs. Independent fusion to different kinds of myosin motor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in the morphology of many animals, the here presented CS diversification reveals many evolutionary complexities. Our findings strongly suggest a very broad and multifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest. PMID:24443419

  10. 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

  11. 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 (csmA?) 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 csmA? 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.

  12. 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

  13. 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

  14. Identification and characterization of two chitin synthase genes in African malaria mosquito, Anopheles gambiae

    PubMed Central

    Zhang, Xin; Zhang, Jianzhen; Park, Yoonseong; Zhu, Kun Yan

    2012-01-01

    Chitin synthase (CHS) represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely tuned chitin biosynthesis and this pathway is absent in humans and other vertebrates. Current knowledge on CHS in insects, especially their structures, functions, and regulations is still very limited. We report the identification and characterization of two chitin synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito, Anopheles gambiae. AgCHS1 and AgCHS2 were predicted to encode proteins of 1,578 and 1,586 amino acid residues, respectively. Their deduced amino acid sequences show high similarities to other insect chitin synthases. Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument. PMID:22683441

  15. 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

  16. 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

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

    PubMed Central

    Sánchez-León, Eddy; Verdín, 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-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (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-1–GFP 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-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex. PMID:21296914

  18. The effects of ergosterol and alcohols on germ-tube formation and chitin synthase in Candida albicans.

    PubMed

    Chiew, Y Y; Sullivan, P A; Shepherd, M G

    1982-01-01

    Ergosterol inhibited germ-tube formation and the membrane-bound enzyme chitin synthase in Candida albicans. The sterol solvents methanol and ethanol stimulated chitin synthase activity but inhibited germination. Methanol increased both the rate of protease activation of the chitin synthase proenzyme and the Vmax of the enzyme. The activation was not due to a change in the protease action on the proenzyme. The methanol activation was reversible and therefore the extraction of inhibitory components from the membrane was not causing the activation. Methanol had no effect on the N-acetylglucosamine activation sites. Analysis of the N-acetylglucosamine activation by an iteration programme gave K1 values of 0.3 mM for the high affinity sites and 20 mM for the low affinity sites. The degree of cooperativity with respect to the substrate uridine diphosphate N-acetylglucosamine was not changed by the presence of methanol and therefore the solvent did not affect the subunit-subunit interactions of the enzyme. Arrhenius plots of chitin synthase have discontinuities and methanol did not affect the transition temperature indicating that the structure of the membrane in the immediate vicinity of the enzyme was unchanged. PMID:6461404

  19. 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...

  20. 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; Grün, 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

  1. 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

  2. Laboratory Evaluation of Five Chitin Synthesis Inhibitors Against the Colorado Potato Beetle, Leptinotarsa decemlineata

    PubMed Central

    Karimzadeh, R.; Hejazi, M. J.; Rahimzadeh Khoei, F.; Moghaddam, M.

    2007-01-01

    Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC50 values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects. PMID:20345285

  3. Novel inhibitors of nitric oxide synthase with antioxidant properties.

    PubMed

    Salerno, Loredana; Modica, Maria N; Romeo, Giuseppe; Pittalà, Valeria; Siracusa, Maria A; Amato, Maria E; Acquaviva, Rosaria; Di Giacomo, Claudia; Sorrenti, Valeria

    2012-03-01

    We previously described a series of imidazole-based inhibitors substituted at N-1 with an arylethanone chain as interesting inhibitors of neuronal nitric oxide synthase (nNOS), endowed with good selectivity vs endothelial nitric oxide synthase (eNOS). As a follow up of these studies, several analogs characterized by the presence of substituted imidazoles or other mono or bicyclic nitrogen-containing heterocycles instead of simple imidazole were synthesized, and their biological evaluation as in vitro inhibitors of both nNOS and eNOS is described herein. Most of these compounds showed improved nNOS and eNOS inhibitory activity with respect to reference inhibitors. Selected compounds were also tested to analyze their antioxidant properties. Some of them displayed good capacity to scavenge free radicals and ability to reduce lipid peroxidation. PMID:22280820

  4. 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.

  5. 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.

  6. Effects of four chitin synthesis inhibitors on feeding and mortality of the Eastern subterranean termite, Reticulitermes flavipes Kollar (Isoptera:Rhinotermitidae) 

    E-print Network

    Vahabzadeh, Rebecca D.

    2002-01-01

    This study measured changes in feeding and mortality of the Eastern subterranean termite, Reticulitermes flavipes when exposed to diets treated with one of four chitin synthesis inhibitors including; diflubenzuron, triflumuron, hexaflumuron...

  7. 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

  8. Design and synthesis of iminosugar-based inhibitors of glucosylceramide synthase: the search for new therapeutic

    E-print Network

    Design and synthesis of iminosugar-based inhibitors of glucosylceramide synthase: the search but not the donor substrate (UDP-glucose) in the glucosylceramide synthase-catalyzed process. Kinetic measure- ments of glucosylceramide synthase, the glucosyltransferase involved in the biosynthesis of glyco- sphingolipids

  9. 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

  10. 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 ?-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 (?Afchs) 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 ?AfchsG mutant was hypersensitive to caspofungin, and all other ?Afchs 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 ?Afchs mutants tested, with the exception of the ?AfchsG 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

  11. 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

  12. 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

  13. 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

  14. Glycogen synthase kinase-3 inhibitors protect central neurons against excitotoxicity.

    PubMed

    Facci, Laura; Stevens, David A; Skaper, Stephen D

    2003-08-01

    Protein kinase B (PKB, or Akt), a downstream effector of phosphatidylinositol 3-kinase (PI-3-K), can play a critical role in regulating neuronal survival. Among known targets of PKB, glycogen synthase kinase-3 (GSK-3) is inhibited by PKB-mediated phosphorylation. Recent studies implicate GSK-3 as a physiologically relevant principal regulatory target of the PI-3-K/PKB survival pathway. Here we show that SB-216763 and SB-415286, selective small molecule inhibitors of GSK-3, protected cultured rat cerebellar granule neurons and hippocampal neurons against excitotoxicity mediated by NMDA and non-NMDA receptor agonists. Treatment with SB-216763 and SB-415286 was optimal when initiated 6-7 days before excitotoxin exposure. As GSK-3 can modulate transcriptional events, these results may provide insight into the identification of new neuroprotective targets. PMID:12960765

  15. The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane.

    PubMed

    Valdivia, Raphael H; Schekman, Randy

    2003-09-01

    During cell stress, Saccharomyces cerevisiae increases the synthesis of chitin and glucans to strengthen and repair the cell wall. In this study, we show that under conditions of cell stress, the steady-state localization of chitin synthase III (Chs3p) shifts from internal stores (chitosomes) to the plasma membrane (PM). This redistribution occurs rapidly and requires the activators of the cell wall stress response signaling pathway, the G protein Rho1p, and the protein kinase Pkc1p, but not the cell integrity response mitogen-activated protein kinase cascade. Furthermore, expression of activated forms of Rho1p or Pkc1p, in the absence of cell stress, is sufficient to redistribute Chs3p to the PM. In cells deficient for both the clathrin adaptor complex 1 and Chs6p, where Chs3p is transported to the PM by an alternative bypass pathway, cell wall stress did not cause mobilization of Chs3p, suggesting that Rho1p/Pkc1p regulate Chs3p exit from the trans-Golgi network. The mobilization of an intracellular reservoir of Chs3p presents a novel opportunity to investigate the genetic basis of regulated vesicular traffic. PMID:12928491

  16. 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

  17. Chitin is endogenously produced in vertebrates.

    PubMed

    Tang, W Joyce; Fernandez, Javier G; Sohn, Joel J; Amemiya, Chris T

    2015-03-30

    Chitin, a biopolymer of N-acetylglucosamine, is abundant in invertebrates and fungi and is an important structural molecule [1, 2]. There has been a longstanding belief that vertebrates do not produce chitin; however, we have obtained compelling evidence to the contrary. Chitin synthase genes are present in numerous fishes and amphibians, and chitin is localized in situ to the lumen of the developing zebrafish gut, in epithelial cells of fish scales, and in at least three different cell types in larval salamander appendages. Chitin synthase gene knockdowns and various histochemical experiments in zebrafish further authenticated our results. Finally, a polysaccharide was extracted from scales of salmon that exhibited all the chemical hallmarks of chitin. Our data and analyses demonstrate the existence of endogenous chitin in vertebrates and suggest that it serves multiple roles in vertebrate biology. PMID:25772447

  18. 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

  19. 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

  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. 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...

  3. ECOLOGICAL FITNESS OF ACETOLACTATE SYNTHASE INHIBITOR–RESISTANT 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) inhibitor–resistant (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. 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

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

    PubMed

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

    2015-03-01

    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 ?-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

  6. 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

  7. 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

  8. Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42.

    PubMed Central

    Semino, C E; Robbins, P W

    1995-01-01

    The Xenopus DG42 gene is expressed only between the late midblastula and neurulation stages of embryonic development. Recent database searches show that DG42 has striking sequence similarity to the Rhizobium NodC protein. NodC catalyzes the synthesis of chitin oligosaccharides which subsequently are transformed into bacterium-plant root signaling molecules. We find that the DG42 protein made in an in vitro coupled transcription-translation system catalyzes the synthesis of an array of chitin oligosaccharides. The result suggests the intriguing possibility that a bacterium-plant type of "Nod" signaling system may operate during early stages of vertebrate embryonic development and raises issues about the use of chitin synthase inhibitors as fungal-specific drugs. Images Fig. 2 PMID:7724589

  9. 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 Å. 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.

  10. 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

  11. 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

  12. Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

    PubMed

    Bartee, David; Morris, Francine; Al-Khouja, Amer; Freel Meyers, Caren L

    2015-08-17

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis. PMID:26174207

  13. Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges

    PubMed Central

    Víte?ek, Jan; Lojek, Antonín; Valacchi, Giuseppe; Kubala, Lukáš

    2012-01-01

    In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted. PMID:22988346

  14. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    SciTech Connect

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-08-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study.

  15. Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase.

    PubMed

    Zhou, Xiang; Ferree, Sarah D; Wills, Veronica S; Born, Ella J; Tong, Huaxiang; Wiemer, David F; Holstein, Sarah A

    2014-05-01

    When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase. PMID:24726306

  16. Geranyl and Neryl Triazole Bisphosphonates as Inhibitors of Geranylgeranyl Diphosphate Synthase

    PubMed Central

    Zhou, Xiang; Ferree, Sarah D.; Wills, Veronica S.; Born, Ella J.; Tong, Huaxiang; Holstein, Sarah A.

    2014-01-01

    When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase. PMID:24726306

  17. Evaluation of Improved Glycogen Synthase Kinase-3? 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 ?-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3? 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-3?/? with the highest GSK-3? selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3? 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-3? inhibition in AML therapy. PMID:26496242

  18. 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

  19. Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.

    PubMed

    Kumar, Vidya P; Frey, Kathleen M; Wang, Yiqiang; Jain, Hitesh K; Gangjee, Aleem; Anderson, Karen S

    2013-10-01

    Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2'-chlorophenyl with a sulfur bridge with a Ki of 8.83±0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors. PMID:23927969

  20. DXP synthase-catalyzed C-N bond formation: Nitroso substrate specificity studies guide selective inhibitor design

    PubMed Central

    Morris, Francine; Vierling, Ryan; Boucher, Lauren; Bosch, Jürgen; Meyers, Caren L. Freel

    2013-01-01

    1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the non-mammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate-dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologs, suggesting it should be pursued as an anti-infective drug target. However, few reports describe development of selective inhibitors of this enzyme. Here, we reveal a function of DXP synthase that catalyzes C-N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP-dependent enzyme Pyruvate Dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate nitroso substrates bind to E. coli DXP synthase in a manner distinct from d-GAP. Our results suggest that incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogs will impart selectivity to DXP synthase inhibitors. As proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP). PMID:23824585

  1. Structure-guided Design of Selective Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Huang, He; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2013-01-01

    Nitric oxide synthases (NOSs) comprise three closely related isoforms that catalyze the oxidation of l-arginine to l-citrulline and the important second messenger nitric oxide (NO). Pharmacological selective inhibition of neuronal NOS (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. Here we present a structure-guided, selective nNOS inhibitor design based on the crystal structure of lead compound 1 in nNOS. The best inhibitor, 7, exhibited low nanomolar inhibitory potency and good isoform selectivities (nNOS over eNOS and iNOS are 472-fold and 239-fold, respectively). Consistent with the good selectivity, 7 binds to nNOS and eNOS with different binding modes. The distinctly different binding modes of 7, driven by the critical residue Asp597 in nNOS, offers compelling insight to explain its isozyme selectivity, which should guide future drug design programs. PMID:23451760

  2. 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

  3. Selective Monocationic Inhibitors of Neuronal Nitric Oxide Synthase. Binding Mode Insights from Molecular Dynamics Simulations

    PubMed Central

    Huang, He; Ji, Haitao; Li, Huiying; Jing, Qing; Labby, Kristin Jansen; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2012-01-01

    The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc. 2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogs. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability. PMID:22731813

  4. Accessible Chiral Linker to Enhance Potency and Selectivity of Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    2013-01-01

    The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily synthesized compounds containing either one (20a,b) or two (9a–d; 15a,b) 2-amino-4-methylpyridine groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor 9c is the best of these compounds, having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS complexed with either 9a or 9c show a double-headed binding mode, where each 2-aminopyridine headgroup interacts with either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of 9c contributes additional hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from the pyrrolidine of 9a to the heme propionate allows the inhibitor to adopt two different binding orientations. Both 9a and 9c bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity. PMID:24660051

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

    PubMed

    Marzinzik, Andreas L; Amstutz, René; Bold, Guido; Bourgier, Emmanuelle; Cotesta, Simona; Glickman, J Fraser; Götte, Marjo; Henry, Christelle; Lehmann, Sylvie; Hartwieg, J Constanze D; Ofner, Silvio; Pellé, Xavier; Roddy, Thomas P; Rondeau, Jean-Michel; Stauffer, Frédéric; 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

  6. 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

  7. 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

  8. Triazolyl phenyl disulfides: 8-Amino-7-oxononanoate synthase inhibitors as potential herbicides.

    PubMed

    Hahn, Hoh-Gyu; Choi, Jung-Sup; Lim, Hee Kyung; Lee, Kee-In; Hwang, In Taek

    2015-11-01

    The chemical validation of a potential herbicide target was investigated with 8-amino-7-oxononanoate synthase (AONS, also known as 7-keto-8-aminopelargonate synthase, KAPAS) and triazolyl phenyl disulfide derivatives in vitro and in vivo. AONS activity was completely inhibited by these synthesized compounds, with an IC50 of 48 to 592?M in vitro. Forty five-day old Arabidopsis thaliana plants were completely killed by representative compound KHG23844 {N-(2-fluorophenyl)-3-(phenyldisulphanyl)-1H-1,2,4-triazole-1-carboxamide} at the application rate of 250gha(-1) of foliar treatment in greenhouse conditions. Foliar application of 1000gha(-1) KHG23844 induced 2.3-fold higher l-alanine accumulation in the treated A. thaliana plants. Foliar supplement of 1mM biotin at 1 and 2days before KHG23844 application effectively recovered the growth inhibition of A. thaliana plant treated with KHG23844. The results strongly suggested that representative compound KHG23844 and its derivatives are potential AONS inhibitors. PMID:26615154

  9. Progress in the development of fatty acid synthase inhibitors as anticancer targets.

    PubMed

    Mullen, Genevieve E; Yet, Larry

    2015-10-15

    Fatty acid synthase (E.C. 2.3.1.85; FASN) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH and plays a central role in lipid biosynthesis. Two classes of FASN exist: FASN I in animals and fungi, and FASN II in plants and prokaryotes. Animal FASN I is a homodimeric protein found in the cytosol of lipogenic tissues such as the liver and brain. Many human carcinomas exhibit elevated levels of FASN I, though the benefit to cancer cells is still unclear. Inhibition of FASN I selectively effects apoptosis in cancer cells, and the role of FASN I in chemotherapy is a growing area of research with the use of natural products and small molecule inhibitors. PMID:26364942

  10. The glucosylceramide synthase inhibitor PDMP sensitizes pancreatic cancer cells to MEK/ERK inhibitor AZD-6244.

    PubMed

    Wang, Ting; Wei, Jue; Wang, Na; Ma, Jia-Li; Hui, Ping-Ping

    2015-01-16

    Here we show that d,l-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a glycosphingolipid biosynthesis inhibitor, increases the sensitivity of pancreatic cancer cells to the novel MEK-ERK inhibitor AZD-6244. AZD-6244 and PDMP co-administration induced massive pancreatic cancer cell death and apoptosis, more potently than either drug alone. We discovered that AZD-6244 induced ceramide production in pancreatic cancer cells, yet the excess ceramide was metabolically removed in the long-term (24-48h). PDMP facilitated AZD-6244-induced ceramide production, and ceramide level remained elevated up to 48h. Meanwhile, exogenously-added cell-permeable short chain ceramide (C2) similarly sensitized AZD-6244's activity, the two caused substantial pancreatic cancer cell death and apoptosis. At the molecular level, PDMP and AZD-6244 co-treatment inactivated ERK1/2 and AKT-mTOR signalings simultaneously in pancreatic cancer cells, while either agent alone only affected one signaling. In summary, PDMP significantly increased the sensitivity of AZD-6244 in pancreatic cancer cells. This appears to involve a sustained ceramide production as well as concurrent block of ERK and AKT-mTOR signalings. PMID:25498501

  11. 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; Haeggström, 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

  12. 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 9.3?M, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1?M. 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

  13. 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

  14. A high-throughput screening assay for identification of inhibitors of the A1AO-ATP synthase of the rumen methanogen Methanobrevibacter ruminantium M1.

    PubMed

    Aung, Htin Lin; Dey, Debjit; Janssen, Peter H; Ronimus, Ron S; Cook, Gregory M

    2015-03-01

    We report the development of a high-throughput screening platform to identify inhibitors of the membrane-bound A1Ao-ATP synthase from the rumen methanogen Methanobrevibacter ruminantium M1. Inhibitors identified in the screen were tested against growing cultures of M. ruminantium, validating the approach to identify new inhibitors of methanogens. PMID:25575416

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

    PubMed

    Broží?ková, C; Mikulecká, A; Otáhal, J

    2014-01-01

    The role of brain derived nitric oxide in the physiology and behavior remains disputable. One of the reasons of the controversies might be systemic side effects of nitric oxide synthase inhibitors. Therefore, under nNOS inhibition by 7-nitroindazole (7-NI) we carried out recordings of blood gasses, blood pressure and spontaneous EEG in conscious adult rats. Locomotion and spontaneous behavior were assessed in an open field. In addition skilled walking and limb coordination were evaluated using a ladder rung walking test. The blood gas analysis revealed a significant increase in pCO(2) 180 min and 240 min after the application of 7-NI. The power and entropy decreased simultaneously with a shift of the mean frequency of the spontaneous EEG toward slow oscillations after 7-NI treatment. The thresholds of evoked potentials underwent a significant drop and a trend towards a slight increase in the I-O curve slope was observed. 7-NI significantly suppressed open field behavior expressed as distance moved, exploratory rearing and grooming. As for the ladder rung walking test the 7-NI treated animals had more errors in foot placement indicating impairment in limb coordination. Therefore our findings suggest that 7-NI increased cortical excitability and altered some physiological and behavioral parameters. PMID:24908089

  1. 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...

  2. 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; ?mura, 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 ?M. 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 ?M, 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

  3. Synthesis and evaluation of C-5 modified 2'-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase.

    PubMed

    Alexandrova, Liudmila A; Chekhov, Vladimir O; Shmalenyuk, Eduard R; Kochetkov, Sergey N; El-Asrar, Rania Abu; Herdewijn, Piet

    2015-11-15

    A series of 5'-monophosphates of 5-substituted 2'-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate 3b, displaying an IC50 value against ThyX of 8.32?M. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX. PMID:26482569

  4. In vitro studies in a myelogenous leukemia cell line suggest an organized binding of geranylgeranyl diphosphate synthase inhibitors.

    PubMed

    Reilly, Jacqueline E; Zhou, Xiang; Tong, Huaxiang; Kuder, Craig H; Wiemer, David F; Hohl, Raymond J

    2015-07-15

    A small set of isoprenoid bisphosphonates ethers has been tested in the K562 chronic myelogenous leukemia cell line to determine their impact on isoprenoid biosynthesis. Five of these compounds inhibit geranylgeranyl diphosphate synthase (GGDPS) with IC50 values below 1 ?M in enzyme assays, but in cells their apparent activity is more varied. In particular, the isomeric C-geranyl-O-prenyl and C-prenyl-O-geranyl bisphosphonates are quite different in their activity with the former consistently demonstrating greater impairment of geranylgeranylation in cells but the latter showing greater impact in the enzyme assays with GGDPS. Together, these findings suggest an organized binding of these inhibitors in the two hydrophobic channels of the geranylgeranyl diphosphate synthase enzyme. PMID:25952057

  5. 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.

  6. 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/?g 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

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

    PubMed

    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

  8. A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro[S

    PubMed Central

    Wasko, Brian M.; Smits, Jacqueline P.; Shull, Larry W.; Wiemer, David F.; Hohl, Raymond J.

    2011-01-01

    Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion. PMID:21903868

  9. 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

  10. 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

  11. 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

  12. Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors

    PubMed Central

    2013-01-01

    Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure–activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11?-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing’s syndrome. PMID:24900631

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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 Alzheime?s disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3? expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3? inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3? 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-3? enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3? 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-3? enzyme activity assays. The inhibitory effect on GSK-3? activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low ?M inhibitor concentrations. Conclusions and Implications: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3? 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

  18. 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.

  19. 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

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

    PubMed Central

    Sprenger, Janina; Svensson, Bo; Hålander, 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

  1. 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, José 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

  2. 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, René; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I.; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V.; Wörheide, 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, 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

  3. 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, René; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V; Wörheide, 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

  4. 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

  5. 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

  6. 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

  7. 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 ?M, 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

  8. 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

  9. Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.

    PubMed

    Symons, Kent T; Nguyen, Phan M; Massari, Mark E; Anzola, John V; Staszewski, Lena M; Wang, Li; Yazdani, Nahid; Dorow, Steven; Muhammad, Jerry; Sablad, Marciano; Rozenkrants, Natasha; Bonefous, Celine; Payne, Joseph E; Rix, Peter J; Shiau, Andrew K; Noble, Stewart A; Smith, Nicholas D; Hassig, Christian A; Zhang, Yan; Rao, Tadimeti S

    2011-02-01

    Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (? 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states. PMID:21036913

  10. Identification of bromophenol thiohydantoin as an inhibitor of DisA, a c-di-AMP synthase, from a 1000 compound library, using the coralyne assay.

    PubMed

    Zheng, Yue; Zhou, Jie; Sayre, David A; Sintim, Herman O

    2014-10-01

    c-di-AMP is an important bacterial second messenger found in Gram-positive and mycobacteria. c-di-AMP regulates myriads of processes in bacteria as well as immune response in higher organisms so interest in small molecules that would attenuate the activity of c-di-AMP metabolism enzymes is high. Herein, we report the first small molecule inhibitor of a c-di-AMP synthase, DisA, using a coralyne-based assay. PMID:25116237

  11. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

    PubMed Central

    Jensen, Samuel A.; Calvert, Andrea E.; Volpert, Giora; Kouri, Fotini M.; Hurley, Lisa A.; Luciano, Janina P.; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H.; Stegh, Alexander H.

    2014-01-01

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

  12. Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica.

    PubMed

    An, Hyo-Jin; Nugroho, Agung; Song, Byong-Min; Park, Hee-Juhn

    2015-01-01

    Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic acid were isolated and identified by NMR spectroscopy. The structure of isoeugenin was determined as 7-hydroxy-5-methoxy-2-methylchromone by the 2D-NMR technique. Among the four compounds, isoeugenin has the lowest IC50 value on the inhibition of NO production in LPS-activated macrophage RAW264.7 cells (IC50, 9.33 ?g/mL). In addition, isoeugenin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines mRNA levels. Taken together, these results suggest that the anti-inflammatory activity of isoeugenin is associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells. Accordingly, our results suggest that the new chromone isoegenin should be considered a potential treatment for inflammatory disease. PMID:26633331

  13. 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.

  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. Identification of 2-mercaptohexanoic acids as dual inhibitors of 5-lipoxygenase and microsomal prostaglandin E? synthase-1.

    PubMed

    Greiner, Christine; Zettl, Heiko; Koeberle, Andreas; Pergola, Carlo; Northoff, Hinnak; Schubert-Zsilavecz, Manfred; Werz, Oliver

    2011-06-01

    5-Lipoxygenase (5-LO) and microsomal prostaglandin E? synthase (mPGES)-1 are key enzymes in the biosynthesis of leukotrienes and prostaglandin (PG)E?, respectively, and are considered as valuable targets for the treatment of inflammatory diseases. Here, we present the identification of 2-mercaptohexanoic acid derivatives as dual inhibitors of 5-LO and mPGES-1. The lead compound 2(4-(3-biphenyloxypropoxy)phenylthio)hexanoic acid (21) inhibits human 5-LO and mPGES-1 in cell-free assays with an IC?? = 3.5 and 2.2 ?M, respectively, and suppresses 5-LO in intact cells with even a higher potency (IC??=0.9 ?M). Compound 21 (10 ?M) neither significantly inhibited the related 12- or 15-LOs nor cyclooxygenase-1 and -2 or cytosolic phospholipase A?. Based on the selective and potent inhibition of 5-LO and mPGES-1, further assessment of these 2-mercaptohexanoic acids in preclinical models of inflammation are warranted. PMID:21570310

  16. 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

  17. Impact of the prostaglandin-synthase 2 inhibitor celecoxib on ovulation and luteal events in women

    PubMed Central

    Edelman, A.B.; Jensen, J.T.; Doom, C; Hennebold, J.D.

    2014-01-01

    Background Ovarian prostaglandins are critical in normal ovulation processes, thus their inhibition may provide contraceptive benefits. This study was performed to determine the effect of the cyclooxygenase-2 (COX2) inhibitor, celecoxib, on ovulation and luteal events in women. Study design Randomized double-blind crossover design. Ovulatory reproductive-aged women underwent ovarian ultrasound and serum hormone monitoring during four menstrual cycles (control cycle, treatment cycle 1, washout cycle, treatment cycle 2). Subjects received study drug (oral celecoxib 400 mg or placebo) either 1) once daily starting on cycle day 8 and continuing until follicle rupture or the onset of next menses if follicle rupture did not occur (pre-LH surge dosing) or 2) once daily beginning with the LH surge and continued for 6 days (post-LH surge dosing). Subjects were randomly assigned to one of the above treatment schemes and received the other in the subsequent treatment cycle. The main outcomes were evidence of ovulatory and luteal dysfunction as determined by inhibited/delayed follicle rupture and reduced luteal progesterone synthesis or lifespan, respectively. Results A total of 20 women enrolled and completed the study (Group 1 = 10, Group 2 = 10) with similar demographics between groups. Nineteen subjects exhibited normal ovulation in the control cycle (one had a blunted LH peak). In comparison to control cycles, treatment cycles resulted in a significant increase in ovulatory dysfunction [pre-LH treatment: 30% (6/20), p = 0.04; post-LH treatment: 25% (5/20), p = 0.04]. Peak progesterone, estradiol, and LH levels and luteal phase length did not differ significantly between control and either treatment cycles. Conclusions Although treatment with celecoxib before or after the LH surge increases the rate of ovulatory dysfunction, most women ovulate normally. Thus, this selective COX2 inhibitor appears to be of limited usefulness as a potential emergency contraceptive. PMID:22902348

  18. 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 (0.043–0.113 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

  19. 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, René; Stelling, Allison; Galli, Roberta; Belikov, Sergei; Petrova, Olga V; Sivkov, Victor V; Vyalikh, Denis; Hunoldt, Sebastian; Wörheide, 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

  20. Inhibitor-?B kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells.

    PubMed

    Natarajan, Mohan; Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L; Mohan, Sumathy

    2015-04-15

    Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-?B kinase-? (IKK?)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKK? on Hsp90. Interestingly, IKK? binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKK? to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKK?. The pathophysiological relevance of the IKK?-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2(Akita) in vivo model. Our study further defines the preferential involvement of ?- vs. ?-isoforms of Hsp90 in the IKK?-eNOS-Hsp90 interaction, even though both Hsp90? and Hsp90? stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKK? within the cell system that regulates NO production, but they also confirm that the IKK?-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

  1. 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

  2. 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 0.05?M trimethoprim, sum of fractional inhibitory concentrations, ?FIC 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

  3. 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

  4. iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3? 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)-3? may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3? 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-3? 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-3? activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3? and its endogenous substrate, glycogen synthase. Results GSK-3? 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-3? activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3? activity in sham-burned rats. Conclusions Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3? activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3?. PMID:22995863

  5. First report on exploring classification and regression based QSAR modelling of Plasmodium falciparum glycogen synthase kinase (PfGSK-3) inhibitors.

    PubMed

    Balasaheb Aher, R; Roy, K

    2015-11-01

    Recent advances in the knowledge of parasite biology have unveiled many new antimalarial targets for drug development. The glycogen synthase kinase-3 from Plasmodium falciparum (PfGSK-3) plays an active role in the completion of the asexual erythrocytic stage of P. falciparum life cycle. Due to the limited availability of experimental information (only one dataset is publicly available to our knowledge) and the absence of the target protein structure, the discovery of new inhibitors against PfGSK-3 is quite challenging. Against this background, we have made an effort to develop classification-based (using linear discriminant analysis or LDA) and regression-based quantitative structure-activity relationship (using group based-QSAR or G-QSAR) models for the categorization and quantitative prediction, respectively, of the activity of PfGSK-3 inhibitors. The classification model highlighted the contribution of electronic (Dipole-mag) and topological (S_tsC) descriptors in discriminating the PfGSK-3 inhibitors into more active and less active classes. The regression-based G-QSAR model showed the contribution of fragment-based descriptors (R1-chiV3 and R2-Most-vePotential) in determining the PfGSK-3 inhibitory activity, and also suggested modification sites for the improvement of PfGSK-3 inhibitory activity. The information obtained from this work could be utilized for the identification of novel PfGSK-3 inhibitors with a hope of overcoming the antimalarial resistance problem. PMID:26524390

  6. Synthesis of isoprenoid bisphosphonate ethers through C-P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase.

    PubMed

    Zhou, Xiang; Reilly, Jacqueline E; Loerch, Kathleen A; Hohl, Raymond J; Wiemer, David F

    2014-01-01

    A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration. PMID:25161722

  7. Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

    PubMed Central

    Zhou, Xiang; Reilly, Jacqueline E; Loerch, Kathleen A; Hohl, Raymond J

    2014-01-01

    Summary A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration. PMID:25161722

  8. 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

  9. Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

    PubMed

    Bortolanza, Mariza; Cavalcanti-Kiwiatkoski, Roberta; Padovan-Neto, Fernando E; da-Silva, Célia Aparecida; Mitkovski, Miso; Raisman-Vozari, Rita; Del-Bel, Elaine

    2015-01-01

    l-3, 4-dihydroxyphenylalanine (l-DOPA) is the most effective treatment for Parkinson's disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of l-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with l-DOPA (30mg/kg plus benserazide 7.5mg/kg, for 21days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with l-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. l-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of l-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinson's disease. PMID:25447229

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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-? 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 TNF? incubation, whereas concentrations of 6-keto PGF1? in supernatants of endothelial cells incubated with TNF? 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 TNF?-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.

  15. Radiosynthesis and preliminary PET evaluation of glycogen synthase kinase 3? (GSK-3?) 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 3? (GSK-3?), 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-3? expression was increased, than in the controls. PMID:26067173

  16. Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress

    PubMed Central

    Moujalled, Diane; James, Janine L.; Parker, Sarah J.; Lidgerwood, Grace E.; Duncan, Clare; Meyerowitz, Jodi; Nonaka, Takashi; Hasegawa, Masato; Kanninen, Katja M.; Grubman, Alexandra; Liddell, Jeffrey R.; Crouch, Peter J.; White, Anthony R.

    2013-01-01

    Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD. PMID:23840699

  17. 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

  18. 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.

  19. Microbial enantioselective ester hydrolysis for the preparation of optically active 4,1-benzoxazepine-3-acetic acid derivatives as squalene synthase inhibitors.

    PubMed

    Tarui, Naoki; Nakahama, Kazuo; Nagano, Yoichi; Izawa, Motowo; Matsumoto, Kiyoharu; Kori, Masakuni; Nagata, Toshiaki; Miki, Takashi; Yukimasa, Hidefumi

    2002-01-01

    Microbial enantioselective ester hydrolysis for the preparation of optically active (3R,5S)-(-)-5-phenyl-4,1-benzoxazepine-3-acetic acid derivatives as potent squalene synthase inhibitors was investigated. Pseudomonas diminuta and Pseudomonas taetrolens hydrolyzed the racemic ethyl ester of the 5-(2-chlorophenyl) analogue to yield the (-)-carboxylic acid with excellent enantiomeric excess (>99% ee). We found that the (-)-enantiomer was an active inhibitor. Bulkiness of the ester moiety did not affect the enantioselectivity but did affect reactivity. The racemic ethyl ester of the 5-(2-methoxyphenyl) analogue, 5-(2,3-dimethoxyphenyl) analogue and 5-(2,4-dimethoxyphenyl) analogue were also hydrolyzed with Pseudomonas taetrolens to afford enantiomerically pure (-)-carboxylic acids in large scale. As another route to (3R,5S)-(-)-7-chloro-5-(2,3-dimethoxyphenyl)-1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid [(-)-1c], the earlier intermediate (-)-2-amino-5-chloro-alpha-(2,3-dimethoxyphenyl)benzyl alcohol [(-)-12] was successfully obtained by asymmetric hydrolysis of (+/-)-5-chloro-alpha-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl acetate with Pseudomonas sp. S-13 with >99% ee in kilogram scale followed by alkaline treatment. The product (-)-12 was converted to (-)-1c without racemization. PMID:11824586

  20. 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

  1. 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

  2. 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

  3. 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; Müller-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

  4. 4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors.

    PubMed

    Maeda, Yutaka; Nakano, Masato; Sato, Hideyuki; Miyazaki, Yasushi; Schweiker, Stephanie L; Smith, Jeffery L; Truesdale, Anne T

    2004-08-01

    Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24. PMID:15225695

  5. 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 {angstrom} 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{beta} (HsGSK-3{beta}) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  6. 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 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-3? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

  7. 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

  8. 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 µM), 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

  9. Inhibitors

    MedlinePLUS

    ... caregivers to learn how to care for the device. CDC Research CDC is interested in learning more about why some people develop inhibitors and how they could be prevented. The Inhibitor Project began in 2005 and was implemented to explore ...

  10. 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

  11. 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

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

    PubMed

    Kaya, Murat; Baublys, Vykintas; Šatkauskien?, Ingrida; Akyuz, Bahar; Bulut, Esra; Tubelyt?, 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 ? 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

  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. 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/?CT imaging. GSK-3 inhibitors caused ?-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/?CT. 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.

  15. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3? inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3? 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-3? over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3? 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-3? and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3?. GSK-3? and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3?. The molecules showed IC50 values ranging from 0.825?M to 1.116?M and were 23- to 57-fold selective for GSK-3?. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825?M. 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 ?-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule. PMID:25064440

  16. Improvement of Tissue Survival of Skin Flaps by 5?-Reductase Inhibitors: Possible Involvement of Nitric Oxide and Inducible Nitric Oxide Synthase

    PubMed Central

    Karimi, Ali Asghar; Ajami, Marjan; Asadi, Yasin; Aboutaleb, Nahid; Gorjipour, Fazel; Malekloo, Roya; Pazoki-Toroudi, Hamidreza

    2015-01-01

    Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5?-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats were randomly allocated into six groups: 1, normal saline topical application; 2, azelaic acid (100 mg/flap); 3, finasteride (1 mg/flap); 4, injection of L-NG-nitroarginine methyl ester (L-NAME) (i.p., 20 mg/kg); 5, L-NAME (20 mg/kg, i.p.) + azelaic acid (100 mg/flap, topical); 6, L-NAME (20 mg/kg, i.p.) + finasteride (1 mg/flap, topical). Tissue survival, level of nitric oxide, and iNOS expression in groups were measured. Results: Our data revealed that azelaic acid and finasteride significantly increased the expression of iNOS protein and nitric oxide (NO) levels in graft tissue (P < 0.05). These increases in iNOS expression and NO level were associated with higher survival of the graft tissue. Conclusion: It appears that alterations of the NO metabolism are implicated in the azelaic acid- and finasteride-mediated survival of the skin flaps. PMID:25864816

  17. The effect of nitric oxide synthase inhibitors on the development of analgesic tolerance to dipyrone in mice.

    PubMed

    Yilmaz, Ibrahim; Ulugol, Ahmet

    2009-01-01

    Recent investigations have shown that, similarly to opioids, tolerance develops to the analgesic effects of nonsteroidal anti-inflammatory drugs (NSAIDs). Nitric oxide has been shown to play an important role in opioid-induced analgesic tolerance; we, therefore, planned to determine if nitric oxide also plays role in the analgesic tolerance to dipyrone, a NSAID. Using the hot-plate test in mice, an analgesic tolerance developed to dipyrone with its 150 and 300 mg/kg intraperitoneal doses after 7 days; no tolerance was observed with its dose of 600 mg/kg. Neither 7-nitroindazole (50 mg/kg, i.p.), a neuronal NOS inhibitor, nor aminoguanidine (30 mg/kg, i.p.), an inducible NOS inhibitor, had any effect on dipyrone-induced analgesic tolerance with doses, which also had no analgesic effect when used alone. Our results show that nitric oxide does not play role in the analgesic tolerance to dipyrone; however, further experiments are required to delineate the mechanisms and to take preventive measures against this problem, which will possibly limit the use of NSAIDs. PMID:19326282

  18. 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.

  19. Oral administration of NO synthase inhibitor failed to promote arteriosclerotic lesions in the aorta and the coronary arteries of rabbits fed cholesterol.

    PubMed

    Nakamura, M; Abe, S; Tanaka, M

    1998-11-01

    We examined whether or not the oral administration of L-nitroarginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, promotes cholesterol-induced arteriosclerosis in the aorta and the coronary artery. Thirty-six male Japanese white rabbits were fed 0.5% cholesterol-containing laboratory chow and randomly assigned to the following three groups: (1) water, (2) 80 microg/ml L-NAME and (3) 400 microg/ml L-NAME in drinking water. The rabbits were fed a 0.5% cholesterol-containing diet for 8 months. During the 8-month period, the concentration of total cholesterol and L-nitroarginine in the serum and the mean blood pressure were measured. The concentration of NO3 in the serum was also measured. After sacrifice, the aortic surface involvement (AI%), the ratio of the thickened intima to the media and the contents of the total cholesterol of the aorta, the maximum % stenosis of the subepicardial large coronary artery, the % frequency of the nearly completely occlusive distal small coronary artery and the area of the myocardial fibrosis were all measured. We found no statistical difference among the three groups regarding the degree of arteriosclerotic lesions of the aorta and of the large coronary artery, and the area of myocardial fibrosis, as well as the serum cholesterol exposure index (the area under the curve of the serum total cholesterol concentration) and the mean blood pressure. However, the serum concentration of L-nitroarginine was approximately 50 and 200 microM/l in groups 2 and 3, respectively. The concentration of NO3 in the serum in group 1 was significantly higher than that in groups 2 and 3. We thus conclude, that the oral administration of L-NAME in the rabbits fed a cholesterol-containing diet for 8 months failed to promote arteriosclerotic lesions in the aorta and the coronary artery, even though the serum concentration of L-nitroarginine increased sufficiently to inhibit NO synthase in the arterial endothelium and the NO3 concentration in the serum decreased in the rabbits given L-NAME. PMID:9863538

  20. Chitin nanofibers: preparations, modifications, and applications.

    PubMed

    Ifuku, Shinsuke; Saimoto, Hiroyuki

    2012-06-01

    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. PMID:22539071

  1. Activity of chitin deacetylase from Colletotrichum gloeosporioides on chitinous substrates.

    PubMed

    Pacheco, Neith; Trombotto, Stéphane; 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 28°C. 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 102×10(3) g mol(-1) and a crystallinity index of ca. 60%. PMID:23688474

  2. Differentiations of chitin content and surface morphologies of chitins extracted from male and female grasshopper species.

    PubMed

    Kaya, Murat; Lelešius, 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

  3. Differentiations of Chitin Content and Surface Morphologies of Chitins Extracted from Male and Female Grasshopper Species

    PubMed Central

    Kaya, Murat; Lelešius, 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; 88.45–95.48% and for commercial chitin; 94.95%. PMID:25635814

  4. 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.

  5. 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

  6. Melanin externalization in Candida albicans depends on cell wall chitin structures.

    PubMed

    Walker, Claire A; Gómez, Beatriz L; Mora-Montes, Héctor M; Mackenzie, Kevin S; Munro, Carol A; Brown, Alistair J P; Gow, Neil A R; Kibbler, Christopher C; Odds, Frank C

    2010-09-01

    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 chs3Delta and chs2Delta chs3Delta mutants but were fully externalized in chs8Delta and chs2Delta chs8Delta 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

  7. 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.

  8. Chitin extraction and characterization from Daphnia magna resting eggs.

    PubMed

    Kaya, Murat; Sargin, Idris; Tozak, Kabil Özcan; Baran, Talat; Erdogan, Sevil; Sezen, Göksal

    2013-10-01

    New application areas for chitin and its derivatives have been extensively investigated and there is a solid, growing demand for new chitin sources. In this present study, chitin content of Daphnia magna resting egg (18-21%) was determined for the first time. FTIR, elemental analysis, TGA, XRD and SEM studies revealed the structural and thermal properties of extracted ?-chitin. This study suggests that D. magna resting eggs can be exploited as an attractive alternative chitin source. PMID:23973492

  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. 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

  11. Dissolution of mechanically milled chitin in high temperature water.

    PubMed

    Aida, Taku Michael; Oshima, Kenji; Abe, Chihiro; Maruta, Ryoma; Iguchi, Masayuki; Watanabe, Masaru; Smith, Richard L

    2014-06-15

    Chitin is high in crystallinity in its natural form and does not dissolve into high temperature water (HTW), which often leads to decomposition reactions such as hydrolysis, deacetylation and dehydration when hydrothermally processed. In this work, we investigated the reactions of mechanically milled chitin in HTW. Mechanical milling pretreatment combined with HTW treatment improved the liquefaction of chitin giving a maximum water soluble fraction of 80%, where the untreated chitin was 55%. The reaction mechanism of the milled and raw chitin in HTW was shown to be different. For milled chitin, the dissolution of chitin occurred during the heating period to supercritical water conditions (400°C) at short reaction times (1 min). Extended reaction time (10 min) led to decomposition products and aromatic char formation. For raw chitin, the dissolution of chitin in HTW did not occur, due to its high crystallinity, so that liquefaction proceeded via decomposition reactions. PMID:24721066

  12. 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.

  13. 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

  14. Nanofibrillar chitin aerogels as renewable base catalysts.

    PubMed

    Tsutsumi, Yoshiyuki; Koga, Hirotaka; Qi, Zi-Dong; Saito, Tsuguyuki; Isogai, Akira

    2014-11-10

    We demonstrate the fabrication of chitin nanofibril aerogels and their successful application as base catalysts for the production of useful chemicals. Squid-pen chitin nanofibrils (ChNF) with primary C2-amine groups on their crystalline surfaces were fabricated into highly porous aerogels with high specific surface areas up to 289 m(2) g(-1) using freeze-drying or a supercritical drying process. The prepared ChNF aerogel was used in the aqueous Knoevenagel-condensation reaction and acted as a highly efficient base catalyst, suggesting that the combination of the nanofibrous aerogel structure and primary C2-amines exposed on the crystalline ChNF surface was effective for continuous flow catalysis. Because the ChNF aerogel can be easily prepared from abundant and renewable chitin present in nature, this strategy is a gateway to promoting and conducting green and sustainable chemistry. PMID:25285573

  15. 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

  16. Naproxen and cromolyn as new glycogen synthase kinase 3? inhibitors for amelioration of diabetes and obesity: an investigation by docking simulation and subsequent in vitro/in vivo biochemical evaluation.

    PubMed

    Motawi, Tarek M K; Bustanji, Yasser; El-Maraghy, Shohda A; Taha, Mutasem O; Al Ghussein, Mohamed A S

    2013-09-01

    Naproxen and cromolyn were investigated as new inhibitors of glycogen synthase kinase-3? (GSK-3?) in an attempt to explain their hypoglycemic properties. Study included simulated docking experiments, in vitro enzyme inhibition assay, and in vivo validations. Both drugs not only were optimally fitted within a GSK-3? binding pocket via several attractive interactions with key amino acids but also exhibited potent in vitro enzymatic inhibitory activities of IC50 1.5 and 2.0 µM for naproxen and cromolyn, respectively. In vivo experiments illustrated that both drugs significantly reduced serum glucose and increased hepatic glycogen- and serum insulin levels in normal and type II diabetic Balb/c mice models. In obese animal model, both drugs exhibited significant reduction in mice weights, serum glucose, and resistin levels along with significant elevation in serum insulin, C-peptide, and adiponectin values. It can be concluded that naproxen and cromolyn are novel GSK-3? inhibitors and can help in management of diabetes and obesity. PMID:23784744

  17. 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.

  18. 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

  19. 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 (150°C) 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.

  20. Aqueous foams stabilized by chitin nanocrystals.

    PubMed

    Tzoumaki, Maria V; Karefyllakis, Dimitris; Moschakis, Thomas; Biliaderis, Costas G; Scholten, Elke

    2015-08-21

    The aim of the present study was to explore the potential use of chitin nanocrystals, as colloidal rod-like particles, to stabilize aqueous foams. Chitin nanocrystals (ChN) were prepared by acid hydrolysis of crude chitin and foams were generated mainly by sonicating the respective dispersions. The foamability of the chitin nanocrystals was evaluated and the resulting foams were assessed for their stability, in terms of foam volume reduction and serum release patterns, during storage. Additionally, the samples were studied with light scattering and optical microscopy in order to explore the bubble size distribution and morphology of the foam. Nanocrystal concentration and charge density was varied to alter the packing of the crystals at the interface. At low concentrations of ChNs, foams were stable against coalescence and disproportionation for a period of three hours, whereas at higher concentrations, the foams were stable for several days. The enhanced stability of foams prepared with ChNs, compared to surfactant-stabilized foams, can be mainly attributed to the irreversible adsorption of the ChNs at the air-water interface, thereby providing Pickering stabilization. Both foam volume and stability of the foam were increased with an increase in ChNs concentration, and at pH values around the chitin's pKa (pH 7.0). Under these conditions, the ChNs show minimal electrostatic repulsion and therefore a higher packing of the nanocrystals is promoted. Moreover, decreased electrostatic repulsion enhances network formation between the ChNs in the aqueous films, thereby providing additional stability by gel formation. Overall, ChNs were proven to be effective in stabilizing foams, and may be useful in the design of Pickering-stabilized food grade foams. PMID:26154562

  1. 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.

  2. 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 ?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

  3. 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

  4. 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

  5. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Inst. of Biomembranes and Bioenergetics, CNR, Bari ; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio; Inst. of Biomembranes and Bioenergetics, CNR, Bari

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  6. 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

  7. Antidepressant-like effect of nitric oxide synthase inhibitors and sildenafil against lipopolysaccharide-induced depressive-like behavior in mice.

    PubMed

    Tomaz, V S; Cordeiro, R C; Costa, A M N; de Lucena, D F; Nobre Júnior, H V; de Sousa, F C F; Vasconcelos, S M M; Vale, M L; Quevedo, J; Macêdo, D

    2014-05-30

    Inflammation, oxidative and nitrosative stress underlie depression being assessed in rodents by the systemic administration of lipopolysacharide (LPS). There is an increasing body of evidence of an involvement of nitric oxide (NO) pathway in depression, but this issue was not investigated in LPS-induced model. Thus, herein we evaluated the effects of NO-pathway-modulating drugs, named aminoguanidine, l-NAME, sildenafil and l-arginine, on the behavioral (forced swimming test [FST], sucrose preference [SPT] and prepulse inhibition [PPI] of the startle) and neurochemical (glutathione [GSH], lipid peroxidation, IL-1?) alterations in the prefrontal cortex, hippocampus and striatum as well as in BDNF levels in the hippocampus 24h after LPS (0.5mg/kg, i.p.) administration, a time-point related to depressive-like behavior. Twenty-four hours post LPS there was an increase in immobility time in the FST, decrease in sucrose preference and PPI levels accompanied by a decrease in GSH levels and an increase in lipid peroxidation, IL-1? and hippocampal BDNF levels suggestive of a depressive-like state. The pretreatment with the NOS inhibitors, l-NAME and aminoguanidine as well as sildenafil prevented the behavioral and neurochemical alterations induced by LPS, although sildenafil and l-NAME were not able to prevent the increase in hippocampal BDNF levels induced by LPS. The iNOS inhibitor, aminoguanidine, and imipramine prevented all behavioral and neurochemical alterations induced by LPS. l-arginine did not prevent the alterations in immobility time, sucrose preference and GSH induced by LPS. Taken together our results show that the NO-cGMP pathway is important in the modulation of the depressive-like alterations induced by LPS. PMID:24662848

  8. 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

  9. 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.

  10. 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 492 nm, respectively, after 48 h 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

  11. 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 (10-120 mg) plus pemetrexed/carboplatin (500 mg/m(2) and 5-6 AUC, respectively) across 8 dose levels every 21 days. 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 40 mg. Eleven dose-limiting toxicities (DLTs) occurred in ten patients. DLTs during LY monotherapy occurred at ?40 mg: 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 80 mg 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 40 mg LY. Conclusions The initial safety profile of LY2090314 was established. MTD LY dose with pemetrexed/carboplatin is 40 mg IV every 3 weeks plus ranitidine. Efficacy of LY plus pemetrexed/carboplatin requires confirmation in randomized trials. PMID:26403509

  12. Effects of the inducible nitric-oxide synthase inhibitor L-N(6)-(1-iminoethyl)-lysine on microcirculation and reactive nitrogen species generation in the kidney following lipopolysaccharide administration in mice.

    PubMed

    Wu, Liping; Mayeux, Philip R

    2007-03-01

    The mortality rate for septic patients with acute renal failure is approximately doubled compared with patients with sepsis alone. Unfortunately, the treatment for sepsis-induced renal failure has advanced little during the last several decades. Because sepsis is often caused by lipopolysaccharide (LPS), a mouse model of LPS challenge was used to study the development of kidney injury. We hypothesized that inducible nitric-oxide synthase (iNOS)-catalyzed nitric oxide production and that generation of reactive nitrogen species (RNS) might play a role in the microcirculatory defect and resulting tubular injury associated with LPS administration. Fluorescent intravital videomicroscopy was used to assess renal peritubular capillary perfusion and document RNS generation by renal tubules in real time. As early as 6 h after LPS administration (10 mg/kg i.p.), RNS generation (rhodamine fluorescence), redox stress [NAD(P)H autofluorescence], and the percentage of capillaries without flow were each significantly increased compared with saline-treated mice (p < 0.05). The generation of RNS was supported by the detection of nitrotyrosine-protein adducts in the kidney using immunohistochemistry. The iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL; 3 mg/kg i.p.) completely blocked the increase in rhodamine fluorescence and NAD(P)H autofluorescence and prevented the capillary defects at 6 h after LPS administration. These results suggest that iNOS-derived RNS is an important contributor to the peritubular capillary perfusion defects and RNS generation that occur during sepsis and emphasize that pharmacological inhibition of iNOS may provide beneficial effects during sepsis by improving renal capillary perfusion and reducing RNS generation in the kidney. PMID:17202403

  13. 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.

  14. 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

  15. 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

  16. Metastability of nematic gels made of aqueous chitin nanocrystal dispersions.

    PubMed

    Tzoumaki, Maria V; Moschakis, Thomas; Biliaderis, Costas G

    2010-01-11

    Chitin nanocrystal aqueous dispersions were prepared by acid hydrolysis of crude chitin from crab shells. The resulting dispersions were studied with small deformation oscillatory experiments and polarized optical microscopy under different conditions of nanocrystal concentration, ionic strength, pH, and temperature. The chitin nanocrystal dispersions exhibited a nematic gel-like behavior with increasing solids concentration. The appearance of nematic-like structures could be explained by the Onsager theory for parallel alignment of anisotropic particles on entropic terms, while the sol-gel transition could be attributed to associative interactions between the chitin nanocrystals. With increasing ionic strength and pH, such associative interactions were enhanced, because the repulsive forces due to the electrostatic charges were reduced and, thus, stronger gels were formed. Heating of the nanocrystal dispersions led to further increases in the storage modulus (G'), which were irreversible upon cooling; the rate of G' increase (dG'/dt) was dependent on temperature. PMID:19947640

  17. 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.

  18. INHIBITOR STUDIES ON MYCOBACTERIUM TUBERCULOSIS MALATE SYNTHASE 

    E-print Network

    Owen, Joshua

    2008-08-03

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) has intensified efforts to discover novel drugs for tuberculosis (TB) treatment. Targeting the persistent state of Mtb, a condition in which Mtb is resistant...

  19. 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

  20. 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 (?-(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

  1. 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

  2. Nanostructured membranes based on native chitin nanofibers prepared by mild process.

    PubMed

    Mushi, Ngesa Ezekiel; Butchosa, Nuria; Salajkova, Michaela; Zhou, Qi; Berglund, Lars A

    2014-11-01

    Procedures for chitin nanofiber or nanocrystal extraction from Crustaceans modify the chitin structure significantly, through surface deacetylation, surface oxidation and/or molar mass degradation. Here, very mild conditions were used to disintegrate chitin fibril bundles and isolate low protein content individualized chitin nanofibers, and prepare nanostructured high-strength chitin membranes. Most of the strongly 'bound' protein was removed. The degree of acetylation, crystal structure as well as length and width of the native chitin microfibrils in the organism were successfully preserved. Atomic force microscopy and scanning transmission electron microscopy, showed chitin nanofibers with width between 3 and 4 nm. Chitin membranes were prepared by filtration of hydrocolloidal nanofiber suspensions. Mechanical and optical properties were measured. The highest data so far reported for nanostructured chitin membranes was obtained for ultimate tensile strength, strain to failure and work to fracture. Strong correlation was observed between low residual protein content and high tensile properties and the reasons for this are discussed. PMID:25129742

  3. Ab initio based conformational study of the crystalline ?-chitin.

    PubMed

    Petrov, Michal; Lymperakis, Liverios; Friák, Martin; Neugebauer, Jörg

    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

  4. 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 Central

    Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O’Connell, Joseph D.; Griner, Sarah; Finer-Moore, Janet S.; Stroud, Robert M.

    2013-01-01

    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 over expressed 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

  5. 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%.

  6. 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/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

  7. A simple procedure for preparing chitin oligomers through acetone precipitation after hydrolysis in concentrated hydrochloric acid.

    PubMed

    Kazami, Nao; Sakaguchi, Masayoshi; Mizutani, Daisuke; Masuda, Tatsuhiko; Wakita, Satoshi; Oyama, Fumitaka; Kawakita, Masao; Sugahara, Yasusato

    2015-11-01

    Chitin oligomers are of interest because of their numerous biologically relevant properties. To prepare chitin oligomers containing 4-6 GlcNAc units [(GlcNAc)4-6], ?- and ?-chitin were hydrolyzed with concentrated hydrochloric acid at 40 °C. The reactant was mixed with acetone to recover the acetone-insoluble material, and (GlcNAc)4-6 was efficiently recovered after subsequent water extraction. Composition analysis using gel permeation chromatography and MALDI-TOF mass spectrometry indicated that (GlcNAc)4-6 could be isolated from the acetone-insoluble material with recoveries of approximately 17% and 21% from the starting ?-chitin and ?-chitin, respectively. The acetone precipitation method is highly useful for recovering chitin oligomers from the acid hydrolysate of chitin. The changes in the molecular size and higher-order structure of chitin during the course of hydrolysis were also analyzed, and a model that explains the process of oligomer accumulation is proposed. PMID:26256353

  8. 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

  9. 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

  10. 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

    2015-01-01

    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. Biosorption of gold from computer microprocessor leachate solutions using chitin.

    PubMed

    Côrtes, Letícia 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 35mgg(-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 20gL(-1) of chitin at 298K for 4h. PMID:26188612

  12. Adsorption of Vibrio parahaemolyticus onto Chitin and Copepods

    PubMed Central

    Kaneko, Tatsuo; Colwell, Rita R.

    1975-01-01

    Vibrio parahaemolyticus was observed to adsorb onto chitin particles and copepods. The efficiency of adsorption was found to be dependent on pH and on the concentration of NaCl and other ions found in seawater. Highest efficiency was observed in water samples collected from Chesapeake Bay and lowest in water from the open sea. V. parahaemolyticus was found to adsorb onto chitin with the highest efficiency of the several bacterial strains tested. Escherichia coli and Pseudomonas fluorescens did not adsorb onto chitin. The adsorption effect is considered to be one of the major factors determining the distribution of this species and affecting the annual cycle of V. parahaemolyticus in the estuarine system. PMID:234715

  13. Studies on taxadiene synthase 

    E-print Network

    Chow, Siew Yin

    2006-08-16

    Taxadiene synthase catalyzes the formation of taxadiene from GGPP, the universal building block of diterpenes. The cyclization of GGPP to taxadiene is generally thought to proceed through a series of monocyclic and bicyclic carbocation intermediates...

  14. 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...

  15. Structure of ?-chitin from Berryteuthis magister and its transformation during whisker preparation and polymerization filling.

    PubMed

    Bogdanova, Olga I; Polyakov, Dmitry K; Streltsov, Dmitry R; Bakirov, Artem V; Blackwell, John; Chvalun, Sergey N

    2016-02-10

    Models for the structures of the ?-chitin-protein complex of native and deproteinized squid pen (Berryteuthis magister) based on SAXS and WAXS data are proposed. Chitin fibrils of 25Å in diameter and persistence length of 1200Å are immersed in protein matrix. Average distance between fibrils is 42Å. Deproteinization of the squid pen led to disappearance of the lateral fibril order stabilized by the protein matrix of the native sample. Swelling in water and acrylic acid resulted in an increase in the chitin 010 d-spacing to 14 and 18Å, respectively. A preparation routine for individual chitin nanofibers of few microns in length and with diameter of 40-60Å has been developed. During exfoliation of the chitin in acrylic acid the degree of acetylation does not change. Chitin-based nanocomposites can be prepared by polymerization of acrylic acid in swelled deproteinized samples which takes place mainly in the interfibrillar space of ?-chitin mainly. PMID:26686179

  16. 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

  17. Characterization of Chitin and Chitosan Molecular Structure in Aqueous Solution

    SciTech Connect

    Franca, Eduardo D.; Lins, Roberto D.; Freitas, Luiz C.; Straatsma, TP

    2008-12-01

    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 ?-chitin nanoparticle, adopt the so-called 2-fold helix with ? and ? 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.

  18. Peptide Induced Crystallization of Calcium Carbonate on Wrinkle Patterned Substrate: Implications for Chitin Formation in Molluscs

    PubMed Central

    Sengupta Ghatak, Anindita; Koch, Marcus; Guth, Christina; Weiss, Ingrid M.

    2013-01-01

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

  19. 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; Szabó, Ildikó; 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

  20. 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.

  1. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    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.

  2. 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

  3. 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

  4. 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

  5. Electrospinning and characterization of chitin nanofibril/polycaprolactone nanocomposite fiber mats.

    PubMed

    Ji, Yali; Liang, Kai; Shen, Xinyuan; Bowlin, Gary L

    2014-01-30

    Nanocomposite fiber mats based on biodegradable polycaprolactone (PCL) and chitin nanofibril (n-chitin) were produced via electrospinning. The morphologies, thermal and mechanical properties as well as surface wettability of the fiber mats were studied by scanning electron microscopy, differential scanning calorimetry analysis, thermogravimetric analysis, dynamic mechanical analysis and static water-contact-angle analysis, respectively. The addition of chitin nanofibrils into PCL resulted in a small change in thermal behavior, but a significant improvement in mechanical properties. Moreover, the surface wettability of electrospun fiber mats transformed from hydrophobicity to hydrophilicity when the chitin nanofibril content was more than 25 wt%. In addition, in vitro cell culture results indicated that the addition of chitin nanofibrils can strongly improve the cellular infiltration and migration confirming that the chitin nanofibril was a good reinforcing as well as bioactive filler for PCL. PMID:24299750

  6. Customizing Properties of ?-Chitin in Squid Pen (Gladius) by Chemical Treatments

    PubMed Central

    Ianiro, Alessandro; Di Giosia, Matteo; Fermani, Simona; Samorì, 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. SbnG, a Citrate Synthase in Staphylococcus aureus

    PubMed Central

    Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; Heinrichs, David E.; Murphy, Michael E. P.

    2014-01-01

    In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

  14. Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase Inhibitors and as Antitumor Agents

    PubMed Central

    Gangjee, Aleem; Zaware, Nilesh; Raghavan, Sudhir; Ihnat, Michael; Shenoy, Satyendra; Kisliuk, Roy L.

    2010-01-01

    Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise and several such clinical trials are currently underway. We have designed, synthesized and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-?) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-? and hTS is better than or close to standards. In a COLO-205 xenograft mouse model one of the analogs significantly decreased tumor growth (TGI = 76% at 35 mg/kg), liver metastases and tumor blood vessels compared to a standard drug and to control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents. PMID:20092323

  15. 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

  16. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. 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 (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  17. Benzo[e]isoindole-1,3-diones as potential inhibitors of glycogen synthase kinase-3 (GSK-3). Synthesis, kinase inhibitory activity, zebrafish phenotype, and modeling of binding mode.

    PubMed

    Zou, Haixia; Zhou, Liyan; Li, Yuanzhen; Cui, Yi; Zhong, Hanbing; Pan, Zhengying; Yang, Zhen; Quan, Junmin

    2010-02-11

    Benzo[e]isoindole-1,3-dione derivatives were synthesized, and the effects on GSK-3beta activity and zebrafish embryo growth were evaluated. A series of derivatives show obvious inhibitory activity against GSK-3beta. The most potent inhibitor, 7,8-dimethoxy-5-methylbenzo[e]isoindole-1,3-dione (8a), shows nanomolar IC(50) and obvious phenotype on zebrafish embryo growth associated with the inhibition of GSK-3beta at low micromolar concentration. The interaction mode between 8a and GSK-3beta was characterized by computational modeling. PMID:20030405

  18. Differential inhibitory effects of protoberberines on sterol and chitin biosyntheses in Candida albicans.

    PubMed

    Park, K S; Kang, K C; Kim, J H; Adams, D J; Johng, T N; Paik, Y K

    1999-05-01

    The anti-Candida potentials of 12 Korean medicinal plants were explored: methanol extracts from Coptis rhizoma and Phellodendron amurense caused significant inhibition of growth of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The predominant active components of the extracts were the protoberberines berberine and palmatine; the most potent inhibition of growth was exhibited by berberine on C. krusei (MIC <4 mg/L) and palmatine on C. parapsilosis (MIC 16 mg/L). Both berberine and palmatine inhibited the in-vivo rate of incorporation of L-[methyl-14C]methionine into C-24 of ergosterol in C. albicans (50% inhibition concentration (IC50 values), 25 microM and 300 microM, respectively); this result suggests that sterol 24-methyl transferase (24-SMT) is one of the cellular targets for the antifungal activity of the protoberberines. In-vitro 24-SMT activity in microsomes from the yeast growth form of C. albicans was inhibited by both berberine (inhibition constant (Ki) 232 microM) and palmatine (Ki 257 microM) in a non-competitive manner; inhibition of 24-SMT was more marked for the mycelial form than for the yeast growth form of this organism. Palmatine inhibited chitin synthase from both the yeast and mycelial growth phases of C. albicans in a non-competitive manner (Ki 780 microM). The effects of protoberberines, extracted from established medicinal plants, on both sterol and cell wall biosyntheses in pathogenic fungi indicate that the potential of these compounds, or their semi-synthetic derivatives, as a novel class of antifungal agents should be investigated more fully. PMID:10382888

  19. Preparation, assessment, and comparison of ?-chitin nano-fiber films with different surface charges

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Jiang, Jie; Liu, Liang; Zheng, Ke; Yu, Shiyuan; Fan, Yimin

    2015-05-01

    Chitin nano-fibers with positive and negative charges have been, respectively, produced from partially deacetylated and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidized ?-chitin. The average diameters and lengths of the TEMPO-oxidized chitin nano-fibers (TOChN) were 14 ± 4.3 and 190 ± 140 nm, respectively, and the average diameters and lengths of the partially deacetylated chitin nano-fibers (DEChN) were 6 ± 1.7 and 320 ± 105 nm, respectively. A partially deacetylated chitin nano-fiber film (DEChN-F), a TEMPO-mediated and oxidized chitin nano-fiber film (TOChN-F), and a composite film (DE-TO-ChN-F) consisting of a combination of the two were prepared by drying the dispersions at 40 °C. The DEChN-F, TOChN-F, and DE-TO-ChN-F all have similar tensile strengths of approximately 90 MPa; however, the chitosan film (Chitosan-F) had a tensile strength of approximately 30 MPa. In addition, TOChN-F and DE-TO-ChN-F have a thermal weight loss at 210 °C, and DEChN-F has a thermal weight loss at 280 °C. DEChN-F was found to have antimicrobial activity with regards to Escherichia coli. Finally, the chitin nano-fiber films could be slightly degraded by cellulase, which provided a novel biological performance of the chitin nano-material.

  20. Fluctuation in physicochemical properties of chitins extracted from different body parts of honeybee.

    PubMed

    Kaya, Murat; Mujtaba, Muhammad; Bulut, Esra; Akyuz, Bahar; Zelencova, Laura; Sofi, Karwan

    2015-11-01

    It is well known that physicochemical properties of chitin are related with the extraction method. Recently, it was revealed that some physicochemical properties of chitin are also related with taxonomical relationship. For the first time in this study, it was tested how these properties of chitin are affected by different body parts of one organism. The chitins were extracted from five different body parts (head, thorax, abdomen, legs and wings) of honeybee. These chitins were physicochemically characterized and differences among these body parts were identified. Highest chitin content was observed in legs (13.25%) while the lowest from thorax (6.79%). The surface morphologies of the isolated chitin structures from five different body parts were analyzed with SEM, as a result, five different types of surface morphologies were recorded. However, three different types of surface morphologies were observed only in abdomen. Maximum degradation temperatures (DTG(max)) of thorax, abdomen, legs and wings were recorded between 359 and 367 °C while DTG(max) value of head chitin was found as 308 °C. PMID:26256318

  1. 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

  2. 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 exhibited excellent elasticity and high mechanical durability. The sponges could efficiently collect organics both on the surface and bottom from the water with the highest 58 times of their own weight absorption capacities through the combination of the particular wettability and great porosity. Furthermore, the biodegradation kinetics of the chitin sponge forecasted that the chitin could be completely biodegraded within 32 days by the microorganisms in the soil. This work provided a new pathway to prepare the chitin-based materials for highly effective removal of oil from water, showing potential application in the pollutant remediation field. PMID:25347002

  3. Surface modification of chitin using ultrasound-assisted and supercritical CO2 technologies for cobalt adsorption.

    PubMed

    Dotto, Guilherme L; Cunha, Jeanine M; Calgaro, Camila O; Tanabe, Eduardo H; Bertuol, Daniel A

    2015-09-15

    Ultrasound-assisted (UA) and supercritical CO2 technologies (SCO2) were used to modify the chitin surface and, improve its adsorption characteristics regarding to cobalt. Chitin, before and after the treatments, was characterized by N2 adsorption isotherms (BET), infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Unmodified and surface modified chitins were used as adsorbents to remove cobalt from aqueous solutions. The adsorption study was performed by equilibrium isotherms and kinetic curves. The chitin particle characteristics, such as, surface area, pore volume and porosity were improved by the UA and SCO2 treatments. The crystallinity index decreased after the UA and SCO2 treatments, and also, intense surface modifications were observed. Langmuir and Freundlich models were adequate to represent the adsorption equilibrium. The maximum adsorption capacities were 50.03, 83.94 and 63.08 mg g(-1) for unmodified chitin, UA surface modified chitin and SCO2 surface modified chitin. The adsorption kinetic curves were well represented by the pseudo-second order model. UA and SCO2 technologies are alternatives to modify the chitin surface and improve its adsorption characteristics. PMID:25880046

  4. Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

    PubMed Central

    Jacquiod, Samuel; Franqueville, Laure; Cécillon, Sébastien; M. Vogel, Timothy; Simonet, Pascal

    2013-01-01

    Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, ?-proteobacteria and ?-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome. PMID:24278158

  5. Hybrid of chitin and humic acid as high performance sorbent for Ni(II)

    NASA Astrophysics Data System (ADS)

    Santosa, Sri Juari; Siswanta, Dwi; Kurniawan, Agusta; Rahmanto, Wasino H.

    2007-11-01

    Hybrid of humic acid (HA) and chitin has been synthesized and the hybrid material (chitin-HA) was then applied as sorbent to adsorb Ni(II). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, according to the procedure recommended by IHSS (International Humic Substances Society). The chitin was isolated from crab shell waste of sea food restaurants through deproteination using NaOH 3.5% (w/v) and followed by removal of inorganic impurities using HCl 1 M. The synthesis of chitin-HA was performed by reacting gelatinous chitin solution in HCl 0.5 M and HA solution in NaOH 0.5 M. Parameters investigated in this work consists of effect of medium acidity on the sorption, sorption rate ( ks) and desorption rate ( kd) constants, Langmuir (monolayer) and Freundlich (multilayer) sorption capacities, and energy ( E) of sorption. The ks and kd were determined according to a kinetic model of first order sorption reaching equilibrium, monolayer sorption capacity ( b) and energy ( E) were determined according to the Langmuir isotherm model, and multilayer sorption capacity ( B) was determined based on the Freundlich isotherm model. Sorption of Ni(II) on both chitin and chitin-HA was maximum at pH 8.0. The kinetic expression resulted from the proposed kinetic model has been shown to be more applicable than the commonly known Lagergren equation obtained from the pseudo-first order sorption model. The application of the proposed model revealed that the presence of HA increased the ks from 0.018 min -1 for chitin to 0.031 min -1 for chitin-HA. As for ks, the value of b was also bigger in the presence of HA, i.e. 7.42 × 10 -5 mol/g for chitin and 9.93 × 10 -5 mol/g for the chitin-HA. Unlike ks and b, the value of E slightly decreased from 23.23 to 21.51 kJ/mol for the absence and presence of HA, respectively. It can also be deduced that the presence of HA on chitin contributed more to the additional layer of Ni(II) sorbed on sorbent. Without HA, B for chitin was only 6.17 times higher than b, while with the presence of HA, the enhancement of the sorption capacity from the multilayer ( B) to the monolayer ( b) was 19.40. The increase of ks, b, B, and the decrease of E would be very benefit in the real application of chitin-HA for the recovery of Ni(II) from aqueous samples.

  6. Chitin based polyurethanes using hydroxyl terminated polybutadiene, part III: surface characteristics.

    PubMed

    Zia, Khalid Mahmood; Zuber, Mohammad; Saif, Muhammad Jawwad; Jawaid, Mohammad; Mahmood, Kashif; Shahid, Muhammad; Anjum, Muhammad Naveed; Ahmad, Mirza Nadeem

    2013-11-01

    Hydroxy terminated polybutadiene (HTPB)-chitin based polyurethanes (PUs) with controlled hydrophobicity were synthesized using HTPB and toluene diisocyanate (TDI). The prepolymer was extended with different mass ratios of chitin and 1,4-butane diol (BDO). The effect of chitin contents in chain extender (CE) proportions on surface properties was studied and investigated. Incorporation of chitin contents into the final PU showed decrease in contact angle value of water drop, water absorption (%) and swelling behavior. The antibacterial activity of the prepared samples was affected by varying the chitin contents in the chemical composition of the final PU. The results demonstrated that the use of prepared material can be suggested as non-absorbable suture. PMID:24120963

  7. Effects of supercritical water and mechanochemical grinding treatments on physicochemical properties of chitin.

    PubMed

    Osada, Mitsumasa; Miura, Chika; Nakagawa, Yuko S; Kaihara, Mikio; Nikaido, Mitsuru; Totani, Kazuhide

    2013-02-15

    This study examined the effects of a combined pretreatment with supercritical water and mechanochemical grinding with a ball mill on the physicochemical properties of chitin and its enzymatic degradation. Following pretreatment with a combination of supercritical water and grinding, chitin had a lower mean molecular weight, a lower crystallinity index, a lower crystallite size, greater d-spacing, weaker hydrogen bonds, and the amide group was more exposed compared with untreated chitin. These properties increased the hydrophilicity of the chitin and enhanced its enzymatic degradation. The N,N'-diacetylchitobiose (GlcNAc)(2) yield after enzymatic degradation of chitin following pretreatment with supercritical water (400 °C, 1 min) and grinding (800 rpm, 10 min) was 93%, compared with 5% without any treatment, 37% with supercritical water pretreatment alone (400 °C, 1 min), and 60% with grinding alone (800 rpm, 30 min). PMID:23399191

  8. Development of a coarse-grained ?-chitin model on the basis of MARTINI forcefield.

    PubMed

    Yu, Zechuan; Lau, Denvid

    2015-05-01

    At nanoscale, atomistic simulation is widely used for investigating crystalline chitin fibers, the structural component for many biological materials. However, the longitudinal dimension of naturally occurring chitin fibers exceeds hundreds of nanometer, beyond the investigation range of all-atom simulation due to the limitation of computational power. Under this context, coarse-grained simulation is a useful alternative that facilitates the investigation of a large system. We develop a coarse-grained model for describing the structural and mechanical properties of ?-chitin. The developed coarse-grained model can reasonably predict these properties. Moreover, this model is consistent with existing coarse-grained force fields for proteins. The present model of ?-chitin possesses good potential and applicability in the investigation of natural chitin-based materials at the length scale of several hundred nanometers. PMID:25914123

  9. Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group.

    PubMed

    Yabushita, Mizuho; Kobayashi, Hirokazu; Kuroki, Kyoichi; Ito, Shogo; Fukuoka, Atsushi

    2015-11-01

    Chitin, a polymer of N-acetylglucosamine units with ?-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2 SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53?% yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70?%. Our process can greatly reduce the use of acid compared to the conventional process. PMID:26538108

  10. Blends of chitin and chitosan with polyamide 66

    SciTech Connect

    Gonzalez, V.; Guerrero, C.

    1996-12-31

    For several years, intense interest has been focused on polymer blends in which both components are synthetic polymers. However, few studies have been made on blends in which one component is chitin (QA), or chitosan (QN), the most abundant natural polymers after cellulose. Its chemical structure, based in partially acetilated {beta}-aminosaccharide units, permits the formation of natural blends with proteins and inorganic salts were the intermolecular hydrogen bonds play an important role. The choice of a partner for these natural polymers was made expecting strong interaction between the two polymers. For this reason, on this work, polyamide 66 (P66), has been chosen.

  11. Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms

    PubMed Central

    Sun, Shuyang; Tay, Qi Xiang Martin; Kjelleberg, Staffan; Rice, Scott A; McDougald, Diane

    2015-01-01

    Association of Vibrio cholerae with chitinous surfaces of zooplankton is important for its persistence in marine environments, as it provides accessibility to nutrients and resistance to stresses. Predation by heterotrophic protists has a major impact on the survival of V. cholerae. V. cholerae forms biofilms as its main defensive strategy, and quorum sensing (QS) additionally regulates the production of antiprotozoal factors. The role of chitin and QS regulation in V. cholerae grazing resistance was investigated by exposing V. cholerae wild-type (WT) and QS mutant biofilms grown on chitin flakes to the bacteriotrophic, surface-feeding flagellate Rhynchomonas nasuta. V. cholerae formed more biofilm biomass on chitin flakes compared with nonchitinous surfaces. The growth of R. nasuta was inhibited by WT biofilms grown on chitin flakes, whereas the inhibition was attenuated in QS mutant biofilms. The chitin-dependent toxicity was also observed when the V. cholerae biofilms were developed under continuous flow or grown on a natural chitin source, the exoskeleton of Artemia. In addition, the antiprotozoal activity and ammonium concentration of V. cholerae biofilm supernatants were quantified. The ammonium levels (3.5?mM) detected in the supernatants of V. cholerae WT biofilms grown on chitin flakes were estimated to reduce the number of R. nasuta by >80% in add-back experiments, and the supernatant of QS mutant biofilms was less toxic owing to a decrease in ammonium production. Transcriptomic analysis revealed that the majority of genes involved in chitin metabolism and chemotaxis were significantly downregulated in QS mutant biofilms when grown on chitin compared with the WT biofilms. PMID:25615438

  12. Microbial responses to chitin and chitosan in oxic and anoxic agricultural soil slurries

    NASA Astrophysics Data System (ADS)

    Wieczorek, A. S.; Hetz, S. A.; Kolb, S.

    2014-06-01

    Microbial degradation of chitin in soil substantially contributes to carbon cycling in terrestrial ecosystems. Chitin is globally the second most abundant biopolymer after cellulose and can be deacetylated to chitosan or can be hydrolyzed to N,N'-diacetylchitobiose and oligomers of N-acetylglucosamine by aerobic and anaerobic microorganisms. Which pathway of chitin hydrolysis is preferred by soil microbial communities is unknown. Supplementation of chitin stimulated microbial activity under oxic and anoxic conditions in agricultural soil slurries, whereas chitosan had no effect. Thus, the soil microbial community likely was more adapted to chitin as a substrate. In addition, this finding suggested that direct hydrolysis of chitin was preferred to the pathway that starts with deacetylation. Chitin was apparently degraded by aerobic respiration, ammonification, and nitrification to carbon dioxide and nitrate under oxic conditions. When oxygen was absent, fermentation products (acetate, butyrate, propionate, hydrogen, and carbon dioxide) and ammonia were detected, suggesting that butyric and propionic acid fermentation, along with ammonification, were likely responsible for anaerobic chitin degradation. In total, 42 different chiA genotypes were detected of which twenty were novel at an amino acid sequence dissimilarity of less than 50%. Various chiA genotypes responded to chitin supplementation and affiliated with a novel deep-branching bacterial chiA genotype (anoxic conditions), genotypes of Beta- and Gammaproteobacteria (oxic and anoxic conditions), and Planctomycetes (oxic conditions). Thus, this study provides evidence that detected chitinolytic bacteria were catabolically diverse and occupied different ecological niches with regard to oxygen availability enabling chitin degradation under various redox conditions on community level.

  13. Microbial responses to chitin and chitosan in oxic and anoxic agricultural soil slurries

    NASA Astrophysics Data System (ADS)

    Wieczorek, A. S.; Hetz, S. A.; Kolb, S.

    2014-02-01

    Chitin is the second most abundant biopolymer in terrestrial ecosystems and is subject to microbial degradation. Chitin can be deacetylated to chitosan or can be hydrolyzed to N,N'-diacetylchitobiose and oligomers of N-acetylglucosamine by aerobic and anaerobic microorganisms. Which pathway of chitin hydrolysis is preferred by soil microbial communities has previously been unknown. Supplementation of chitin stimulated microbial activity under oxic and anoxic conditions in agricultural soil slurries, whereas chitosan had no effect. Thus, the soil microbial community likely was more adapted to chitin as a substrate. In addition, this finding suggested that direct hydrolysis of chitin was preferred to the pathway that starts with deacetylation. Chitin was apparently degraded by aerobic respiration, ammonification, and nitrification to carbon dioxide and nitrate under oxic conditions. When oxygen was absent, fermentation products (acetate, butyrate, propionate, hydrogen, carbon dioxide) and ammonia were detected, suggesting that butyric and propionic acid fermentation were along with ammonification likely responsible for apparent anaerobic chitin degradation. In total, 42 different chiA genotypes were detected of which twenty were novel at an amino acid sequence dissimilarity of >50%. Various chiA genotypes responded to chitin supplementation and affiliated with a novel deep-branching bacterial chiA genotype (anoxic conditions), genotypes of Beta- and Gammaproteobacteria (oxic and anoxic conditions), and Planctomycetes (oxic conditions). Thus, this study provides evidence that detected chitinolytic bacteria were catabolically diverse and occupied different ecological niches with regard to oxygen availability enabling chitin degradation under various redox conditions at the level of the community.

  14. Structures of human constitutive nitric oxide synthases.

    PubMed

    Li, Huiying; Jamal, Joumana; Plaza, Carla; Pineda, Stephanie Hai; Chreifi, Georges; Jing, Qing; Cinelli, Maris A; Silverman, Richard B; Poulos, Thomas L

    2014-10-01

    Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03?Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73?Å resolution. PMID:25286850

  15. Angiogenesis Inhibitors

    MedlinePLUS

    ... inhibitors currently being used to treat cancer in humans? How are angiogenesis inhibitors different from conventional anticancer ... inhibitors currently being used to treat cancer in humans? Yes. The U.S. Food and Drug Administration (FDA) ...

  16. Activation of enzymatic chitin degradation by a lytic polysaccharide monooxygenase.

    PubMed

    Hamre, Anne Grethe; Eide, Kristine B; Wold, Hanne H; Sørlie, Morten

    2015-04-30

    For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent k(cat) (k(cat)(app)) values on a ?-chitin substrate. k(cat)(app) values were determined to be 1.7±0.1 s(-1) and 1.7±0.1 s(-1) for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s(-1) for the endo-active ChiC. The addition of CBP21 boosted the k(cat)(app) values of ChiA and ChiB giving values of 11.1±1.5 s(-1) and 13.9±1.4 s(-1), while there was no effect on ChiC (0.9±0.1 s(-1)). PMID:25812992

  17. Applying Molecular Dynamics Simulations to Identify Rarely Sampled Ligand-bound Conformational States of Undecaprenyl Pyrophosphate Synthase, an Antibacterial Target

    SciTech Connect

    Sinko, William; de Oliveira, César; Williams, Sarah; Van Wynsberghe, Adam; Durrant, Jacob D.; Cao, Rong; Oldfield, Eric; McCammon, J. Andrew

    2012-04-30

    Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentally validated undecaprenyl pyrophosphate synthase inhibitors using high- and low-populated conformational states extracted from the molecular dynamics simulations, we show that structurally dissimilar compounds can bind preferentially to different and rarely sampled conformational states. By performing structural analyses on the newly obtained apo-undecaprenyl pyrophosphate synthase and other crystal structures previously published, we show that the changes observed during the molecular dynamics simulation are very similar to those seen in the crystal structures obtained in the presence or absence of ligands. We believe that this is the first time that a rare 'expanded pocket' state, key to drug design and verified by crystallography, has been extracted from a molecular dynamics simulation.

  18. Description of a new surface morphology for chitin extracted from wings of cockroach (Periplaneta americana).

    PubMed

    Kaya, Murat; Baran, Talat

    2015-04-01

    In this study a new morphology of chitin, which could find wide applications in the fields of medicine, pharmacy, agriculture, food and textiles, has been described. The chitin was isolated from the wings of Periplaneta americana employing a conventional method. Considering chitin isolation studies conducted previously, chitin has three surface morphologies, which are (1) hard and rough surface without pores or nanofibers, (2) surface solely composed of nanofibers and (3) surfaces with both pores and nanofibers. In this study, the surface of the chitin, examined with environmental scanning electron microscopy (ESEM), only has oval nanopores (230-510 nm) without nanofibers, and this is different from the above mentioned surface morphologies. The nanopores are not distributed on the chitin surface randomly. Typically, there is a pore in the center that is surrounded by six or seven other pores in an ordered manner. Structures similar to cell walls exist between the pores. Chitin with the new surface morphology was characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction (XRD) and elemental analysis. PMID:25597430

  19. Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin.

    PubMed

    Peters, W; Latka, I

    1986-01-01

    The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microscopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5-10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthogonal texture of bundles of microfibrils that are thought to contain chitin. The presence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues. PMID:3754855

  20. Green Conversion of Agroindustrial Wastes into Chitin and Chitosan by Rhizopus arrhizus and Cunninghamella elegans Strains

    PubMed Central

    Berger, Lúcia Raquel Ramos; Stamford, Thayza Christina Montenegro; Stamford-Arnaud, Thatiana Montenegro; de Alcântara, Sergio Roberto Cabral; da Silva, Antonio Cardoso; da Silva, Adamares Marques; do Nascimento, Aline Elesbão; de Campos-Takaki, Galba Maria

    2014-01-01

    This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin and chitosan were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, viscosity and X-ray diffraction. The effectiveness of chitosan from C. elegans and R. arrhizus in inhibiting the growth of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Yersinia enterocolitica were evaluated by determining the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The highest production of biomass (24.60 g/L), chitin (83.20 mg/g) and chitosan (49.31 mg/g) was obtained by R. arrhizus. Chitin and chitosan from both fungi showed a similar degree of deacetylation, respectively of 25% and 82%, crystallinity indices of 33.80% and 32.80% for chitin, and 20.30% and 17.80% for chitosan. Both chitin and chitosan presented similar viscosimetry of 3.79–3.40 cP and low molecular weight of 5.08 × 103 and 4.68 × 103 g/mol. They both showed identical MIC and MBC for all bacteria assayed. These results suggest that: agricultural wastes can be produced in an environmentally friendly way; chitin and chitosan can be produced economically; and that chitosan has antimicrobial potential against pathogenic bacteria. PMID:24853288

  1. Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection.

    PubMed

    Hoseini, Mostafa Haji Molla; Moradi, Maryam; Alimohammadian, Mohammad Hossein; Shahgoli, Vahid Khaze; Darabi, Hayedeh; Rostami, Ali

    2016-04-01

    Chitin and chitosan microparticles (MPs) are important immune system stimulators. The aim of this study was to evaluate the protective effects of these compounds in comparison with each other against Leishmania infection in BALB/c mice infected with Leishmania major (L. major). Female BALB/c mice were injected subcutaneously with 2×10(5) promastigotes. Chitin and/or chitosan MPs (<40?m) were subcutaneously injected in the BALB/c mice with two-day intervals until two weeks. Mice in all groups were sacrificed at 12weeks post-infection. Enumeration of viable parasites was performed using limiting dilution assay. Furthermore, the animals (5 mice/group) were sacrificed two weeks post-infection. The lymph node cells were isolated and the effects of the chitinous MPs on the proliferation and production of cytokines such as tumor necrosis factor alpha (TNF-?) and interleukin-10 (IL-10) were determined. The mean sizes of lesions were significantly smaller in chitin (0.6±0.12mm) and chitosan treated groups (1.2±0.8mm) than in the control group (6.2±1.7mm) (P<0.05). The parasite load in the lymph nodes of the treated mice was significantly lower than that in the lymph nodes of controls (1.31×10(6) vs 8.24×10(7) parasite/lymph node [P=0.032] and 7.49×10(6) vs 8.24×10(7) parasite/lymph node [P=0.05] for chitin and chitosan MPs treatment, respectively). We found that chitinous MPs induced cell proliferation and that chitin but not chitosan increased TNF-? and IL-10 production. Chitin appears that it has more effect than chitosan against leishmaniasis. The current study revealed that chitinous MPs had significant activity against L. major and could be considered as new therapeutic modality in leishmaniasis. PMID:26518128

  2. Effects of chitin and its derivatives on human cancer cells lines.

    PubMed

    Bouhenna, M; Salah, R; Bakour, R; Drouiche, N; Abdi, N; Grib, H; Lounici, H; Mameri, N

    2015-10-01

    The present study is focused on the effect of chitin derivatives against human cancer cell lines RD and Hep2. As an outcome from this research, chitin was cytotoxic at IC50?=?400 ?g/ml and 200 ?g/ml against Hep2 cells and RD cells lines, respectively. Irradiated chitin had an IC50 value of 450 ?g/ml for Hep2 and an IC50 of 200 ?g/ml for RD. The lowest IC50 is attributed to chitosan, 300 ?g/ml in Hep2 and 190 ?g/ml in RD. PMID:26013739

  3. Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta

    PubMed Central

    Ehrlich, H.; Rigby, J. Keith; Botting, J. P.; Tsurkan, M. V.; Werner, C.; Schwille, P.; Petrášek, Z.; Pisera, A.; Simon, P.; Sivkov, V. N.; Vyalikh, D. V.; Molodtsov, S. L.; Kurek, D.; Kammer, M.; Hunoldt, S.; Born, R.; Stawski, D.; Steinhof, A.; Bazhenov, V. V.; Geisler, T.

    2013-01-01

    Sponges are probably the earliest branching animals, and their fossil record dates back to the Precambrian. Identifying their skeletal structure and composition is thus a crucial step in improving our understanding of the early evolution of metazoans. Here, we present the discovery of 505–million-year-old chitin, found in exceptionally well preserved Vauxia gracilenta sponges from the Middle Cambrian Burgess Shale. Our new findings indicate that, given the right fossilization conditions, chitin is stable for much longer than previously suspected. The preservation of chitin in these fossils opens new avenues for research into other ancient fossil groups. PMID:24336573

  4. The role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases.

    PubMed

    Mack, Ines; Hector, Andreas; Ballbach, Marlene; Kohlhäufl, Julius; Fuchs, Katharina J; Weber, Alexander; Mall, Marcus A; Hartl, Dominik

    2015-12-01

    Chitin, after cellulose, the second most abundant biopolymer on earth, is a key component of insects, fungi, and house-dust mites. Lower life forms are endowed with chitinases to defend themselves against chitin-bearing pathogens. Unexpectedly, humans were also found to express chitinases as well as chitinase-like proteins that modulate immune responses. Particularly, increased levels of the chitinase-like protein YKL-40 have been associated with severe asthma, cystic fibrosis, and other inflammatory disease conditions. Here, we summarize and discuss the potential role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases. PMID:26542293

  5. Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta.

    PubMed

    Ehrlich, H; Rigby, J Keith; Botting, J P; Tsurkan, M V; Werner, C; Schwille, P; Petrášek, Z; Pisera, A; Simon, P; Sivkov, V N; Vyalikh, D V; Molodtsov, S L; Kurek, D; Kammer, M; Hunoldt, S; Born, R; Stawski, D; Steinhof, A; Bazhenov, V V; Geisler, T

    2013-01-01

    Sponges are probably the earliest branching animals, and their fossil record dates back to the Precambrian. Identifying their skeletal structure and composition is thus a crucial step in improving our understanding of the early evolution of metazoans. Here, we present the discovery of 505-million-year-old chitin, found in exceptionally well preserved Vauxia gracilenta sponges from the Middle Cambrian Burgess Shale. Our new findings indicate that, given the right fossilization conditions, chitin is stable for much longer than previously suspected. The preservation of chitin in these fossils opens new avenues for research into other ancient fossil groups. PMID:24336573

  6. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    SciTech Connect

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  7. Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates.

    PubMed

    Vattekkatte, Abith; Gatto, Nathalie; Schulze, Eva; Brandt, Wolfgang; Boland, Wilhelm

    2015-04-28

    The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies. PMID:25807032

  8. MICROBIAL FERMENTATION OF ABUNDANT BIOPOLYMERS: CELLULOSE AND CHITIN

    SciTech Connect

    Leschine, Susan

    2009-10-31

    Our research has dealt with seven major areas of investigation: i) characterization of cellulolytic members of microbial consortia, with special attention recently given to Clostridium phytofermentans, a bacterium that decomposes cellulose and produces uncommonly large amounts of ethanol, ii) investigations of the chitinase system of Cellulomonas uda; including the purification and characterization of ChiA, the major component of this enzyme system, iii) molecular cloning, sequence and structural analysis of the gene that encodes ChiA in C. uda, iv) biofilm formation by C. uda on nutritive surfaces, v) investigations of the effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes, vi) studies of nitrogen metabolism in cellulolytic anaerobes, and vii) understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. Also, progress toward completing the research of more recent projects is briefly summarized. Major accomplishments include: 1. Characterization of Clostridium phytofermentans, a cellulose-fermenting, ethanol-producing bacterium from forest soil. The characterization of a new cellulolytic species isolated from a cellulose-decomposing microbial consortium from forest soil was completed. This bacterium is remarkable for the high concentrations of ethanol produced during cellulose fermentation, typically more than twice the concentration produced by other species of cellulolytic clostridia. 2. Examination of the use of chitin as a source of carbon and nitrogen by cellulolytic microbes. We discovered that many cellulolytic anaerobes and facultative aerobes are able to use chitin as a source of both carbon and nitrogen. This major discovery expands our understanding of the biology of cellulose-fermenting bacteria and may lead to new applications for these microbes. 3. Comparative studies of the cellulase and chitinase systems of Cellulomonas uda. Results of these studies indicate that the chitinase and cellulase systems of this bacterium are distinct in terms of the proteins involved and the regulation of their production. 4. Characterization of the chitinase system of C. uda. A 70,000-Mr endochitinase, designated ChiA, was purified from C. uda culture supernatant fluids and characterized. 5. Analysis of chiA, which codes for the major enzymatic component of the chitinase system of C. uda. The gene encoding the endochitinase ChiA in C. uda was cloned, its complete nucleotide sequence was determined and its implications were investigated. 6. Formation of biofilms by C. uda on cellulose and chitin. Microscopic observations indicated that, under conditions of nitrogen limitation, C. uda cells grew as a biofilm attached tightly to the surface of cellulose or chitin. 7. Development of tools for a genetic approach to studies of cellulose fermentation by cellulolytic clostridia. We have explored the potential of various techniques, and obtained evidence indicating that Tn916 mutagenesis may be particularly effective in this regard. As part of this research, we identified the presence of a plasmid in one strain, which was cloned, sequenced, and analyzed for its utility in the development of vectors for genetic studies. 8. Effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes. We determined that humic substances play an important role in the anaerobic cellulose decomposition and in the physiology of cellulose-fermenting soil bacteria. 9. Nitrogenases of cellulolytic clostridia. We described a nitrogenase gene from a cellulolytic clostridium and presented evidence, based on sequence analyses and conserved gene order, for lateral gene transfer between this bacterium and a methanogenic archaeon. 10. Characterization of Clostridium hungatei, a new N2-fixing cellulolytic species isolated from a methanogenic consortium from soil. 11. Understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. We discovered that C. papyrosolvens produces a multiprotein, multicom

  9. The Pursuit of Unshocking Hsp90 Inhibitors: Development of Gedunin and Cruentaren A as Chemical Leads

    E-print Network

    Brandt, Gary Edward

    2011-12-31

    of Gedunin Analogues ----------------------------------------- 65 Chapter III Total Synthesis of Cruentaren A, Selective F1Fo ATP Synthase Inhibitor Isolated from Byssovorax cruenta Table 3.1: The Warburg Effect...

  10. Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient.

    PubMed

    Tan, YerPeng; Hoon, Shawn; Guerette, Paul A; Wei, Wei; Ghadban, Ali; Hao, Cai; Miserez, Ali; Waite, J Herbert

    2015-07-01

    The beak of the jumbo squid Dosidicus gigas is a fascinating example of how seamlessly nature builds with mechanically mismatched materials. A 200-fold stiffness gradient begins in the hydrated chitin of the soft beak base and gradually increases to maximum stiffness in the dehydrated distal rostrum. Here, we combined RNA-Seq and proteomics to show that the beak contains two protein families. One family consists of chitin-binding proteins (DgCBPs) that physically join chitin chains, whereas the other family comprises highly modular histidine-rich proteins (DgHBPs). We propose that DgHBPs play multiple key roles during beak bioprocessing, first by forming concentrated coacervate solutions that diffuse into the DgCBP-chitin scaffold, and second by inducing crosslinking via an abundant GHG sequence motif. These processes generate spatially controlled desolvation, resulting in the impressive biomechanical gradient. Our findings provide novel molecular-scale strategies for designing functional gradient materials. PMID:26053298

  11. Characteristics of a Streptomyces coelicolor A3(2) Extracellular Protein Targeting Chitin and Chitosan

    PubMed Central

    Saito, Akihiro; Miyashita, Kiyotaka; Biukovi?, Goran; Schrempf, Hildgund

    2001-01-01

    Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets ?-chitin, ?-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described. PMID:11229920

  12. Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan

    PubMed Central

    Kumirska, Jolanta; Czerwicka, Ma?gorzata; Kaczy?ski, Zbigniew; Bychowska, Anna; Brzozowski, Krzysztof; Thöming, Jorg; Stepnowski, Piotr

    2010-01-01

    Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds. PMID:20559489

  13. Chitin-mediated plant-fungal interactions: catching, hiding and handshaking.

    PubMed

    Shinya, Tomonori; Nakagawa, Tomomi; Kaku, Hanae; Shibuya, Naoto

    2015-08-01

    Plants can detect infecting fungi through the perception of chitin oligosaccharides by lysin motif receptors such as CEBiP and CERK1. A major function of CERK1 seems to be as a signaling molecule in the receptor complex formed with ligand-binding molecules and to activate downstream defense signaling. Fungal pathogens, however, have developed counter strategies to escape from the chitin-mediated detection by using effectors and/or changing their cell walls. Common structural features between chitin and Nod-/Myc-factors and corresponding receptors have suggested the close relationships between the chitin-mediated immunity and rhizobial/arbuscular mycorrhizal symbiosis. The recent discovery of the dual function of OsCERK1 in both plant immunity and mycorrhizal symbiosis sheds new light on the evolutionary relationships between defense and symbiotic systems in plants. PMID:26116978

  14. Biomineralization of calcium phosphate crystals on chitin nanofiber hydrogel for bone regeneration material.

    PubMed

    Kawata, Mari; Azuma, Kazuo; Izawa, Hironori; Morimoto, Minoru; Saimoto, Hiroyuki; Ifuku, Shinsuke

    2016-01-20

    We previously reported a chitin nanofiber hydrogel from squid pen ?-chitin by a simple NaOH treatment. In the present study, a calcium phosphate/chitin nanofiber hydrogel was prepared for bone tissue engineering. Calcium phosphate was mineralized on the hydrogel by incubation in a solution of diammonium hydrogen phosphate solution followed by calcium nitrate tetrahydrate. X-ray diffractometry and Fourier transform infrared spectroscopy showed the formation of calcium phosphate crystals. The morphology of the calcium phosphate crystals changed depending on the calcification time. After mineralization, the mechanical properties of the hydrogel improved due to the reinforcement effect of calcium phosphate crystal. In an animal experiment, calcium phosphate/chitin nanofiber hydrogel accelerated mineralization in subcutaneous tissues. Morphological osteoblasts were observed. PMID:26572435

  15. Chitin, Chitosan, and Glycated Chitosan Regulate Immune Responses: The Novel Adjuvants for Cancer Vaccine

    PubMed Central

    Li, Xiaosong; Min, Min; Du, Nan; Gu, Ying; Hode, Tomas; Naylor, Mark; Chen, Dianjun; Nordquist, Robert E.; Chen, Wei R.

    2013-01-01

    With the development of cancer immunotherapy, cancer vaccine has become a novel modality for cancer treatment, and the important role of adjuvant has been realized recently. Chitin, chitosan, and their derivatives have shown their advantages as adjuvants for cancer vaccine. In this paper, the adjuvant properties of chitin and chitosan were discussed, and some detailed information about glycated chitosan and chitosan nanoparticles was also presented to illustrate the trend for future development. PMID:23533454

  16. Improved colorimetric determination of cell wall chitin in wood decay fungi.

    PubMed

    Chen, G C; Johnson, B R

    1983-07-01

    The Svennerholm modification of the Elson-Morgan method for glucosamine analysis was evaluated for its applicability to the rapid determination of chitin in wood decay fungi. The evaluation included extent of chromogen interference, sensitivity, color stability, and hydrolysis conditions for maximum release of glucosamine from fungal cell walls. With our further modification, the Svennerholm method was shown to be suitable for rapid quantitative determination of fungal chitin without chromatographic separation of hydrolysate chromogens. PMID:16346335

  17. Formulation development of stronger and quick disintegrating tablets: a crucial effect of chitin.

    PubMed

    Goel, Honey; Kaur, Gurpreet; Tiwary, Ashok K; Rana, Vikas

    2010-05-01

    A well known superdisintegrant like croscarmellose sodium or crospovidone loses their quick disintegration property when compressed at more than 4 kg tablet crushing strength (TCS). Therefore, the objective of the present work was to develop a disintegrating system that could be used for preparing fast disintegrating tablets (FDTs) of highly water soluble drug, metoclopramide, without compromising on the mechanical strength, irrespective of the TCS used for compressing the granules. For this purpose disintegrating system consisting of chitosan-alginate (CTN-ALG) complex (1:1): glycine and chitin was developed. The results revealed that when CTN-ALG and glycine were mixed in the ratio of 30:70, the granules exhibited a minimum water sorption time and maximum effective pore radius (R(eff.p)). The addition of chitin (5-10% w/w) into this mixture further enhanced the R(eff.p). Further, increase in the concentration of chitin from 10% to 20% w/w did not produce any significant effect (p>0.05) on the R(eff.p). The FDTs prepared by using CTN-ALG:glycine (30:70) and chitin exhibited increased porosity and lower disintegration time (DT). Further, chitin was found to neutralize the effect of TCS on DT of FDTs. This property of chitin was also observed in FDTs prepared by using croscarmellose sodium (5% w/w) or crospovidone (5% w/w). The reduction in DT of FDTs by chitin was also observed in tablets prepared without the drug. Hence, the effect was not influenced by the solubility component present in the tablets. Overall, the results suggested incorporation of chitin (5-10% w/w) while preparing FDTs of metoclopramide to enhanced the disintegration without compromising their mechanical strength of the tablets. PMID:20460872

  18. Chitin and chitosan from Brazilian Atlantic Coast: Isolation, characterization and antibacterial activity.

    PubMed

    Abdel-Rahman, Rasha M; Hrdina, Radim; Abdel-Mohsen, A M; Fouda, Moustafa M G; Soliman, A Y; Mohamed, F K; Mohsin, Kazi; Pinto, Tiago Dinis

    2015-09-01

    Chitin and chitosan were obtained by chemical treatments of shrimp shells. Different particle sizes (50-1000 ?m) of the raw material were used to study their effect on size distribution, demineralization, deproteinization and deacetylation of chitin and chitosan isolation process. The particle size in the range of 800-1000 ?m was selected to isolate chitin, which was achieved by measuring nitrogen, protein, ash, and yield %. Hydrochloric acid (5%, v/v) was optimized in demineralization step to remove the minerals from the starting material. Aqueous solution of sodium hydroxide (5%, w/v) at 90 °C for (20 h) was used in deproteinization step to remove the protein. Pure chitin was consequently impregnated into high concentration of sodium hydroxide (50%) for 3.5 h at 90 °C to remove the acetyl groups in order to form high pure chitosan. The degree of deacetylation (DDA) of chitosan was controlled and evaluated by different analytical tools. The chemical structure of chitin and chitosan was confirmed by elemental analysis, ATR-FTIR, H/C NMR, XRD, SEM, UV-Vis spectroscopy, TGA, and acid-base titration. The isolated chitin and chitosan from shrimp shell showed excellent antibacterial activity against Gram (-ve) bacteria (Escherichia coli) comparing with commercial biopolymers. PMID:26093316

  19. Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

    PubMed Central

    Madhuprakash, Jogi; El Gueddari, Nour Eddine; Moerschbacher, Bruno M.; Podile, Appa Rao

    2015-01-01

    Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on ?- and ?-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP. PMID:25615694

  20. Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri

    PubMed Central

    Schaefer, Amy L.; Brennan, Caitlin A.; Heath-Heckman, Elizabeth A. C.; DeLoney-Marino, Cindy R.; McFall-Ngai, Margaret J.

    2012-01-01

    Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae (“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone. PMID:22522684

  1. Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels.

    PubMed

    Huang, Yao; Yao, Mengyu; Zheng, Xing; Liang, Xichao; Su, Xiaojuan; Zhang, Yu; Lu, Ang; Zhang, Lina

    2015-11-01

    Novel nanocomposite hydrogels composed of polyelectrolytes alginate and chitin whiskers with biocompatibility were successfully fabricated based on the pH-induced charge shifting behavior of chitin whiskers. The chitin whiskers with mean length and width of 300 and 20 nm were uniformly dispersed in negatively charged sodium alginate aqueous solution, leading to the formation of the homogeneous nanocomposite hydrogels. The experimental results indicated that their mechanical properties were significantly improved compared to alginate hydrogel and the swelling trends were inhibited as a result of the strong electrostatic interactions between the chitin whiskers and alginate. The nanocomposite hydrogels exhibited certain crystallinity and hierarchical structure with nanoscale chitin whiskers, similar to the structure of the native extracellular matrix. Moreover, the nanocomposite hydrogels were successfully applied as bone scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs and scanning electronic microscope (SEM) images revealed that the addition of chitin whiskers into the nanocomposite hydrogels markedly promoted the cell adhesion and proliferation of the osteoblast cells. The biocompatible nanocomposite hydrogels have potential application in bone tissue engineering. PMID:26393272

  2. The effect of chitin metabolic effectors on the population increase of stored product mites.

    PubMed

    Stara, Jitka; Erban, Tomas; Hubert, Jan

    2010-10-01

    The study tested the effect of the chitin metabolic effectors, teflubenzuron, diflubenzuron, and calcofluor, and a combination of a chitinase and soybean trypsin inhibitor (STI) on the population growth of eight species of stored product mites under laboratory conditions. The compounds were incorporated into the diets of the mites in concentrations ranging from 0.01 to 50 mg g(-1). The final populations of mites were observed after 21 days of growth in controlled conditions. Diflubenzuron and calcofluor suppressed the growth of all the tested species more effectively than the other compounds. The doses required to suppress the mite populations to 50% (rc(50)) in comparison to the control ranged from 0.29 to 12.68 mg g(-1) for diflubenzuron and from 1.75 to 37.7 mg g(-1) for calcofluor, depending on the mite species. When tested at the highest concentration (10 mg g(-1)), teflubenzuron also suppressed all of the tested mite species in comparison to the control. The addition of chitinase/STI into the diet influenced population growth in several ways. When the highest concentration of chitinase in a cocktail of chitinase and STI (12.5 mg g(-1) of diet) was compared to the control, populations of Acarus siro, Aleuroglyphus ovatus and Aëroglyphus robustus decreased significantly, whereas populations of Tyroborus lini and Sancassania rodionovi increased significantly. The sensitivity of species to the tested compounds differed among species. The most tolerant species was S. rodionovi, the most sensitive was A. ovatus. The results confirmed that calcofluor and diflubenzuron have a toxic effect on stored product mites. PMID:20229097

  3. Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle.

    PubMed

    Reggiani, R; Nebuloni, M; Mattana, M; Brambilla, I

    2000-01-01

    Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8h of anaerobic treatment and, by immunoprecipitation of 35S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation. PMID:10901618

  4. Synthesis and utilization of chitin humic acid hybrid as sorbent for Cr(III)

    NASA Astrophysics Data System (ADS)

    Santosa, Sri Juari; Siswanta, Dwi; Sudiono, Sri; Sehol, Muhamad

    2007-11-01

    New types of hybrid material have been synthesized by using four different methods of immobilization of humic acid (HA) on chitin. The most stable hybrid material toward the change of medium acidity was then utilized as sorbent for Cr(III). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, using the recommended procedure of International Humic Substances Society (IHSS), while the chitin was isolated from crab shell waste through deproteination using 3.5% (w/v) NaOH and followed by removal of inorganic impurities using 1 M HCl. The four methods of immobilization of HA on chitin were (i) Method A: chitin powder (4 g) was gently poured into the stirred solution of 0.4 g HA in 40 mL of 0.01 M NaOH. After overnight stirring, the solid was separated, washed with water, and dried in oven at 70 °C. (ii) Method B: gelatinous chitin (40 g) in 250 mL of 0.5 M HCl was reacted with HA (4 g) in 500 mL of 0.5 M NaOH and aged for 24 h. The product was washed with water and dried. (iii) Method C: HA powder (0.5 g) was mixed with the stirred gel of chitin (2.5 g) in 60 mL of CaCl 2 saturated methanol and the mixture was then washed with the mixed solution of 25 mL of 2 M sodium citrate and ethylene glycol 1:1. The solid was separated, washed with water, and dried. (iv) Method D: the solution of HA (0.056 g) in 10 mL of 0.01 M NaOH was reacted with the gel of chitin (0.2 g) in 10 mL of CaCl 2 saturated methanol. After 24 h stirring, the solid was separated from the reaction medium, washed with the mixed solution of 2 M sodium citrate and ethylene glycol 1:1, and followed by washing with water and drying. Parameters investigated in this study consisted of the stability test of the immobilized HA, as well as the rate constant ( k1), capacity ( b), and energy ( E) of sorption as well as the rate constant of desorption ( k-1). The k1 and k-1 were determined according to a kinetic model of first order sorption reaching equilibrium, while the b and E were determined according to the Langmuir isotherm model. Compared to HA, Methods, A, C, and D; Method B produced the most stable immobilization of HA on chitin. The hybrid material (Chitin-HA) synthesized through Method B was stable in the acidity range that equivalent to pH 2.0-11.0. At the acidity giving maximum sorption, i.e. pH 5, the presence of immobilized HA on the Chitin-HA enhanced more than three times the k1 and k-1, i.e. from 0.057 min -1 and 8.51 × 10 -4 (min -1) (mol/L) for chitin to 0.183 min -1 and 3.27 × 10 -3 (min -1) (mol/L) for the Chitin-HA. On the contrary, the presence of HA on Chitin-HA only gave small increase on b and small decrease on E. The values of b and E for Cr(III) on chitin were 1.45 × 10 -2 mol/g and 23.12 kJ/mol, respectively, while those on Chitin-HA were 1.78 × 10 -2 mol/g and 19.95 kJ/mol, respectively.

  5. Keeping track of the growing number of biological functions of chitin and its interaction partners in biomedical research.

    PubMed

    Koch, Bjørn E V; Stougaard, Jens; Spaink, Herman P

    2015-05-01

    Chitin is a vital polysaccharide component of protective structures in many eukaryotic organisms but seems absent in vertebrates. Chitin or chitin oligomers are therefore prime candidates for non-self-molecules, which are recognized and degraded by the vertebrate immune system. Despite the absence of polymeric chitin in vertebrates, chitinases and chitinase-like proteins (CLPs) are well conserved in vertebrate species. In many studies, these proteins have been found to be involved in immune regulation and in mediating the degradation of chitinous external protective structures of invading pathogens. Several important aspects of chitin immunostimulation have recently been uncovered, advancing our understanding of the complex regulatory mechanisms that chitin mediates. Likewise, the last few years have seen large advances in our understanding of the mechanisms and molecular interactions of chitinases and CLPs in relation to immune response regulation. It is becoming increasingly clear that their function in this context is not exclusive to chitin producing pathogens, but includes bacterial infections and cancer signaling as well. Here we provide an overview of the immune signaling properties of chitin and other closely related biomolecules. We also review the latest literature on chitinases and CLPs of the GH18 family. Finally, we examine the existing literature on zebrafish chitinases, and propose the use of zebrafish as a versatile model to complement the existing murine models. This could especially be of benefit to the exploration of the function of chitinases in infectious diseases using high-throughput approaches and pharmaceutical interventions. PMID:25595947

  6. Adsorption of Silver Nanoparticles onto Different Surface Structures of Chitin/Chitosan and Correlations with Antimicrobial Activities

    PubMed Central

    Ishihara, Masayuki; Nguyen, Vinh Quang; Mori, Yasutaka; Nakamura, Shingo; Hattori, Hidemi

    2015-01-01

    Size-controlled spherical silver nanoparticles (Ag NPs) can be simply prepared by autoclaving mixtures of glass powder containing silver with glucose. Moreover, chitins with varying degrees of deacetylation (DDAc < 30%) and chitosan powders and sheets (DDAc > 75%) with varying surface structure properties have been evaluated as Ag NP carriers. Chitin/chitosan-Ag NP composites in powder or sheet form were prepared by mixing Ag NP suspensions with each of the chitin/chitosan-based material at pH 7.3, leading to homogenous dispersion and stable adsorption of Ag NPs onto chitin carriers with nanoscale fiber-like surface structures, and chitosan carriers with nanoscale porous surface structures. Although these chitins exhibited mild antiviral, bactericidal, and antifungal activities, chitin powders with flat/smooth film-like surface structures had limited antimicrobial activities and Ag NP adsorption. The antimicrobial activities of chitin/chitosan-Ag NP composites increased with increasing amounts of adsorbed Ag NPs, suggesting that the surface structures of chitin/chitosan carriers strongly influence adsorption of Ag NPs and antimicrobial activities. These observations indicate that chitin/chitosan-Ag NPs with nanoscale surface structures have potential as antimicrobial biomaterials and anti-infectious wound dressings. PMID:26096004

  7. Adsorption of Silver Nanoparticles onto Different Surface Structures of Chitin/Chitosan and Correlations with Antimicrobial Activities.

    PubMed

    Ishihara, Masayuki; Nguyen, Vinh Quang; Mori, Yasutaka; Nakamura, Shingo; Hattori, Hidemi

    2015-01-01

    Size-controlled spherical silver nanoparticles (Ag NPs) can be simply prepared by autoclaving mixtures of glass powder containing silver with glucose. Moreover, chitins with varying degrees of deacetylation (DDAc < 30%) and chitosan powders and sheets (DDAc > 75%) with varying surface structure properties have been evaluated as Ag NP carriers. Chitin/chitosan-Ag NP composites in powder or sheet form were prepared by mixing Ag NP suspensions with each of the chitin/chitosan-based material at pH 7.3, leading to homogenous dispersion and stable adsorption of Ag NPs onto chitin carriers with nanoscale fiber-like surface structures, and chitosan carriers with nanoscale porous surface structures. Although these chitins exhibited mild antiviral, bactericidal, and antifungal activities, chitin powders with flat/smooth film-like surface structures had limited antimicrobial activities and Ag NP adsorption. The antimicrobial activities of chitin/chitosan-Ag NP composites increased with increasing amounts of adsorbed Ag NPs, suggesting that the surface structures of chitin/chitosan carriers strongly influence adsorption of Ag NPs and antimicrobial activities. These observations indicate that chitin/chitosan-Ag NPs with nanoscale surface structures have potential as antimicrobial biomaterials and anti-infectious wound dressings. PMID:26096004

  8. Structural requirements of synthetic and natural product lipo-chitin oligosaccharides for induction of nodule primordia on Glycine soja.

    PubMed Central

    Stokkermans, T J; Ikeshita, S; Cohn, J; Carlson, R W; Stacey, G; Ogawa, T; Peters, N K

    1995-01-01

    Rhizobia synthesize a class of lipo-chitin oligosaccharides that induce root hair deformation and induce the initiation of nodule structures on legume roots. These lipo-chitin oligosaccharides are tetra- and penta-lipo-oligosaccharides of N-acetylglucosamine with an acyl substitution on the nonreducing end and are commonly known as Nod factors. In this study, we demonstrate that synthetic analogs of natural product Nod factors have the same biological activities. To determine structure-activity relationships, a collection of synthetic and natural product lipo-chitin oligosaccharides was assayed on Glycine soja. All biologically active lipo-chitin oligosaccharides induced both root hair deformation and nodule initiations on G. soja. The most active lipo-chitin oligosaccharides deformed root hairs at 10(-15) M and induced nodules at 1 ng of lipo-chitin oligosaccharide per spot inoculation. Plant responses demonstrate an interdependence of backbone length and the presence of substitutions on the reducing end. Lipo-chitin oligosaccharides containing four N-acetylglucosamine residues were active only without a reducing end modification, whereas lipo-chitin oligosaccharides containing five N-acetylglucosamine residues were active only with reducing end modification. The plant thus recognizes lipo-chitin oligosaccharides without reducing end substitutions despite the importance of these modifications for host range. PMID:7659753

  9. Genetics Home Reference: GM3 synthase deficiency

    MedlinePLUS

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... synthase deficiency and may include treatment providers. American Epilepsy Society: Find a Doctor Clinic for Special Children ( ...

  10. Promiscuous archaeal ATP synthase concurrently coupled to Na+ translocation

    E-print Network

    Huang, Ching-Tsan

    Promiscuous archaeal ATP synthase concurrently coupled to Na+ and H+ translocation Katharina98B02057 Chii-Shen Yang, PhD December 25, 2012 The 6th Classroom ATP (adenosine triphosphate) synthase ATP ATP ATP synthase ATP synthase ATP synthase ATP synthase Methanosarcina acetivorans

  11. Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.

    PubMed Central

    Moldes, M; Boizard, M; Liepvre, X L; Fève, B; Dugail, I; Pairault, J

    1999-01-01

    We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes. PMID:10585876

  12. Interfacing whispering gallery mode optical microresonator biosensors with the plant defense elicitor chitin.

    PubMed

    Dahmen, Jeremy L; Yang, Yongqiang; Greenlief, C Michael; Stacey, Gary; Hunt, Heather K

    2014-10-01

    The biomaterial class of chitooligosaccharides (chitin), commonly found in insects and fungi, is one of the most abundant on earth. Substantial evidence implicates chitin in mediating a diverse array of plant cellular signaling events, including the induction of plant defense mechanisms against invading pests. However, these recognition and mediation mechanisms, including the binding kinetics between chitin and their plant recognition receptors, are not fully understood. Therefore, the creation of a platform capable of both interfacing with chitin and plant cell receptors, and monitoring their interactions, would significantly advance our understanding of this plant defense elicitor. Recently, a label-free, highly sensitive biosensor platform, based on Whispering Gallery Mode optical microresonators, has been developed to study such biomolecular interactions. Here, we demonstrate how this unique platform can be interfaced with chitin using simple carbohydrate chemistry. The surface chemistry is demonstrated using X-ray photoelectron spectroscopy, fluorescence microscopy, optical profilometry, ellipsometry, and contact angle measurements. The resulting surface is uniform, with an average surface roughness of 1.25nm, and is active toward chitin recognition elements. Optical loss measurements using standard quantitative cavity analysis techniques demonstrate that the bioconjugated platforms maintain the high performance (Q>10(6)) required to track binding interactions in this system. The platform is able to detect lectin, which binds COs, at 10?g/mL concentration. This biosensor platform's unique capabilities for label-free, high sensitivity biodetection, when properly interfaced with the biomaterials of interest, could provide the basis for a robust analytical technique to probe the binding dynamics of chitin-plant cell receptors. PMID:25051306

  13. Synthesis of GTMAC modified chitin-PAA gel and evaluation of its biological properties.

    PubMed

    Uppanan, Paweena; Channasanon, Somruethai; Veeranondh, Sukitaya; Tanodekaew, Siriporn

    2011-08-01

    The dressing prepared from GTMAC modified chitin-PAA was introduced with the aim of facilitating wound healing, particularly those effectively absorbing exudates, maintaining a moist wound environment and controlling bacterial proliferation. Chitin was chemically modified with acrylic acid to encourage a moist wound healing environment. The highly water-absorbable resulting product (chitin-PAA) was further reacted with glycidyltrimethylammonium chloride (GTMAC) to impart antibacterial activities. The final product, chitin-PAA-GTMAC was characterized by the techniques of Fourier Transform Infrared (FTIR), solid state (15) N NMR, and elemental analysis. Their cytotoxicity and antibacterial activities against S. epidermidis and E. coli were evaluated which found increasing effects in those properties with increasing degree substitution of GTMAC. All materials also showed good blood-clotting ability. The collagen gel contraction assay was used to analyze the behavior of fibroblasts after contact with the gels. The extent of the gel contraction as well as the examination of the secreted interleukin-8 (IL-8) and transforming growth factor-?1 (TGF-?1) were investigated. The results showed that chitin-PAA modified with GTMAC could stimulate the production of IL-8, but TGF-?1. Fibroblasts presented normal spreading and formation of cellular processes in the collagen gels with all of the modifications. Furthermore, all modified gels except for the highest GTMAC content gel [chitin-PAA-GTMAC (1:20)] were found a greater extent in gel contraction than the unmodified chitin-PAA. It suggested the promoting effect of GTMAC on cell proliferation if the GTMAC content in the gel was not too high, that is, the mole ratio of glucosamine to GTMAC of the gel should not greater than 1:10. PMID:21548069

  14. Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites.

    PubMed

    Ozer, Ufuk; Barbour, Karen W; Clinton, Sarah A; Berger, Franklin G

    2015-12-01

    Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming dTMP for the maintenance of DNA replication and repair. Inhibitors of TYMS have been widely used in the treatment of neoplastic disease. A number of fluoropyrimidine and folate analogs have been developed that lead to inhibition of the enzyme, resulting in dTMP deficiency and cell death. In the current study, we have examined the role of oxidative stress in response to TYMS inhibitors. We observed that intracellular reactive oxygen species (ROS) concentrations are induced by these inhibitors and promote apoptosis. Activation of the enzyme NADPH oxidase (NOX), which catalyzes one-electron reduction of O2 to generate superoxide (O2 (?-)), is a significant source of increased ROS levels in drug-treated cells. However, gene expression profiling revealed a number of other redox-related genes that may contribute to ROS generation. TYMS inhibitors also induce a protective response, including activation of the transcription factor nuclear factor E2-related factor 2 (NRF2), a critical mediator of defense against oxidative and electrophilic stress. Our results show that exposure to TYMS inhibitors induces oxidative stress that leads to cell death, while simultaneously generating a protective response that may underlie resistance against such death. PMID:26443810

  15. Chemical inhibitors of monogalactosyldiacylglycerol synthases in Arabidopsis thaliana.

    PubMed

    Botté, Cyrille Y; Deligny, Michael; Roccia, Aymeric; Bonneau, Anne-Laure; Saïdani, Nadia; Hardré, Hélène; Aci, Samia; Yamaryo-Botté, Yoshiki; Jouhet, Juliette; Dubots, Emmanuelle; Loizeau, Karen; Bastien, Olivier; Bréhélin, Laurent; Joyard, Jacques; Cintrat, Jean-Christophe; Falconet, Denis; Block, Maryse A; Rousseau, Bernard; Lopez, Roman; Maréchal, Eric

    2011-11-01

    Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main lipids in photosynthetic membranes in plant cells. They are synthesized in the envelope surrounding plastids by MGD and DGD galactosyltransferases. These galactolipids are critical for the biogenesis of photosynthetic membranes, and they act as a source of polyunsaturated fatty acids for the whole cell and as phospholipid surrogates in phosphate shortage. Based on a high-throughput chemical screen, we have characterized a new compound, galvestine-1, that inhibits MGDs in vitro by competing with diacylglycerol binding. Consistent effects of galvestine-1 on Arabidopsis thaliana include root uptake, circulation in the xylem and mesophyll, inhibition of MGDs in vivo causing a reduction of MGDG content and impairment of chloroplast development. The effects on pollen germination shed light on the contribution of galactolipids to pollen-tube elongation. The whole-genome transcriptional response of Arabidopsis points to the potential benefits of galvestine-1 as a unique tool to study lipid homeostasis in plants. PMID:21946275

  16. Squid pen chitin chitooligomers as food colorants absorbers.

    PubMed

    Liang, Tzu-Wen; Huang, Chih-Ting; Dzung, Nguyen Anh; Wang, San-Lang

    2015-01-01

    One of the most promising applications of chitosanase is the conversion of chitinous biowaste into bioactive chitooligomers (COS). TKU033 chitosanase was induced from squid pen powder (SPP)-containing Bacillus cereus TKU033 medium and purified by ammonium sulfate precipitation and column chromatography. The enzyme was relatively more thermostable in the presence of the substrate and had an activity of 93% at 50 °C in a pH 5 buffer solution for 60 min. Furthermore, the enzyme used for the COS preparation was also studied. The enzyme products revealed various mixtures of COS that with different degrees of polymerization (DP), ranging from three to nine. In the culture medium, the fermented SPP was recovered, and it displayed a better adsorption rate (up to 96%) for the disperse dyes than the water-soluble food colorants, Allura Red AC (R40) and Tartrazne (Y4). Fourier transform-infrared spectroscopic (FT-IR) analysis proved that the adsorption of the dyes onto fermented SPP was a physical adsorption. Results also showed that fermented SPP was a favorable adsorber and could be employed as low-cost alternative for dye removal in wastewater treatment. PMID:25608726

  17. Squid Pen Chitin Chitooligomers as Food Colorants Absorbers

    PubMed Central

    Liang, Tzu-Wen; Huang, Chih-Ting; Dzung, Nguyen Anh; Wang, San-Lang

    2015-01-01

    One of the most promising applications of chitosanase is the conversion of chitinous biowaste into bioactive chitooligomers (COS). TKU033 chitosanase was induced from squid pen powder (SPP)-containing Bacillus cereus TKU033 medium and purified by ammonium sulfate precipitation and column chromatography. The enzyme was relatively more thermostable in the presence of the substrate and had an activity of 93% at 50 °C in a pH 5 buffer solution for 60 min. Furthermore, the enzyme used for the COS preparation was also studied. The enzyme products revealed various mixtures of COS that with different degrees of polymerization (DP), ranging from three to nine. In the culture medium, the fermented SPP was recovered, and it displayed a better adsorption rate (up to 96%) for the disperse dyes than the water-soluble food colorants, Allura Red AC (R40) and Tartrazne (Y4). Fourier transform-infrared spectroscopic (FT-IR) analysis proved that the adsorption of the dyes onto fermented SPP was a physical adsorption. Results also showed that fermented SPP was a favorable adsorber and could be employed as low-cost alternative for dye removal in wastewater treatment. PMID:25608726

  18. Injectable Amorphous Chitin-Agarose Composite Hydrogels for Biomedical Applications.

    PubMed

    Priya, Murali Vishnu; Kumar, Rajendran Arun; Sivashanmugam, Amirthalingam; Nair, Shantikumar Vasudevan; Jayakumar, Rangasamy

    2015-01-01

    Injectable hydrogels are gaining popularity as tissue engineering constructs because of their ease of handling and minimal invasive delivery. Making hydrogels from natural polymers helps to overcome biocompatibility issues. Here, we have developed an Amorphous Chitin (ACh)-Agarose (Agr) composite hydrogel using a simpletechnique. Rheological studies, such as viscoelastic behavior (elastic modulus, viscous modulus, yield stress, and consistency), inversion test, and injectability test, were carried out for different ACh-Agr concentrations. The composite gel, having a concentration of 1.5% ACh and 0.25% Agr, showed good elastic modulus (17.3 kPa), yield stress (3.8 kPa), no flow under gravity, injectability, and temperature stability within the physiological range. Based on these studies, the optimum concentration for injectability was found to be 1.5% ACh and 0.25% Agr. This optimized concentration was used for further studies and characterized using FT-IR and SEM. FT-IR studies confirmed the presence of ACh and Agr in the composite gel. SEM results showed that the lyophilized composite gel had good porosity and mesh like networks. The cytocompatibility of the composite gel was studied using human mesenchymal stem cells (hMSCs). The composite gels showed good cell viability.These results indicated that this injectable composite gel can be used for biomedical applications. PMID:26308065

  19. Injectable Amorphous Chitin-Agarose Composite Hydrogels for Biomedical Applications

    PubMed Central

    Priya, Murali Vishnu; Kumar, Rajendran Arun; Sivashanmugam, Amirthalingam; Nair, Shantikumar Vasudevan; Jayakumar, Rangasamy

    2015-01-01

    Injectable hydrogels are gaining popularity as tissue engineering constructs because of their ease of handling and minimal invasive delivery. Making hydrogels from natural polymers helps to overcome biocompatibility issues. Here, we have developed an Amorphous Chitin (ACh)-Agarose (Agr) composite hydrogel using a simpletechnique. Rheological studies, such as viscoelastic behavior (elastic modulus, viscous modulus, yield stress, and consistency), inversion test, and injectability test, were carried out for different ACh-Agr concentrations. The composite gel, having a concentration of 1.5% ACh and 0.25% Agr, showed good elastic modulus (17.3 kPa), yield stress (3.8 kPa), no flow under gravity, injectability, and temperature stability within the physiological range. Based on these studies, the optimum concentration for injectability was found to be 1.5% ACh and 0.25% Agr. This optimized concentration was used for further studies and characterized using FT-IR and SEM. FT-IR studies confirmed the presence of ACh and Agr in the composite gel. SEM results showed that the lyophilized composite gel had good porosity and mesh like networks. The cytocompatibility of the composite gel was studied using human mesenchymal stem cells (hMSCs). The composite gels showed good cell viability.These results indicated that this injectable composite gel can be used for biomedical applications. PMID:26308065

  20. Chitin butyrate coated electrospun nylon-6 fibers for biomedical applications

    NASA Astrophysics Data System (ADS)

    Pant, Hem Raj; Kim, Han Joo; Bhatt, Lok Ranjan; Joshi, Mahesh Kumar; Kim, Eun Kyo; Kim, Jeong In; Abdal-hay, Abdalla; Hui, K. S.; Kim, Cheol Sang

    2013-11-01

    In this study, we describe the preparation and characterizations of chitin butyrate (CB) coated nylon-6 nanofibers using single-spinneret electrospinning of blends solution. The physicochemical properties of nylon-6 composite fibers with different proportions of CB to nylon-6 were determined using FE-SEM, TEM, FT-IR spectroscopy, and water contact angle measurement. FE-SEM and TEM images revealed that the nylon-6 and CB were immiscible in the as-spun nanofibers, and phase separated nanofiber morphology becomes more pronounced with increasing amounts of CB. The bone formation ability of composite fibers was evaluated by incubating in biomimetic simulated body fluid. In order to assay the cytocompatibility and cell behavior on the composite scaffolds, osteoblast cells were seeded on the matrix. Results suggest that the deposition of CB layer on the surface of nylon-6 could increase its cell compatibility and bone formation ability. Therefore, as-synthesized nanocomposite fibrous mat has great potentiality in hard tissue engineering.

  1. Modification of Chitin with Kraft Lignin and Development of New Biosorbents for Removal of Cadmium(II) and Nickel(II) Ions

    PubMed Central

    Wysokowski, Marcin; Klapiszewski, ?ukasz; Moszy?ski, Dariusz; Bartczak, Przemys?aw; Szatkowski, Tomasz; Majchrzak, Izabela; Siwi?ska-Stefa?ska, Katarzyna; Bazhenov, Vasilii V.; Jesionowski, Teofil

    2014-01-01

    Novel, functional materials based on chitin of marine origin and lignin were prepared. The synthesized materials were subjected to physicochemical, dispersive-morphological and electrokinetic analysis. The results confirm the effectiveness of the proposed method of synthesis of functional chitin/lignin materials. Mechanism of chitin modification by lignin is based on formation of hydrogen bonds between chitin and lignin. Additionally, the chitin/lignin materials were studied from the perspective of waste water treatment. The synthetic method presented in this work shows an attractive and facile route for producing low-cost chitin/lignin biosorbents with high efficiency of nickel and cadmium adsorption (88.0% and 98.4%, respectively). The discovery of this facile method of synthesis of functional chitin/lignin materials will also have a significant impact on the problematic issue of the utilization of chitinous waste from the seafood industry, as well as lignin by-products from the pulp and paper industry. PMID:24727394

  2. Chitin-glucan complex production by Komagataella pastoris: Downstream optimization and product characterization.

    PubMed

    Farinha, Inês; Duarte, Paulo; Pimentel, Ana; Plotnikova, Evgeniya; Chagas, Bárbara; Mafra, Luís; Grandfils, Christian; Freitas, Filomena; Fortunato, Elvira; Reis, Maria A M

    2015-10-01

    Purified chitin-glucan complex (CGCpure) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a ?-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGCpure had an average molecular weight of 4.9 × 10(5)Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGCpure has ?-chitin in its structure. CGCpure presented an endothermic decomposition peak at 315°C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products. PMID:26076647

  3. Thin-film Nanofibrous Composite Membranes Containing Cellulose or Chitin Barrier Layers Fabricated by Ionic Liquids

    SciTech Connect

    H Ma; B Hsiao; B Chu

    2011-12-31

    The barrier layer of high-flux ultrafiltration (UF) thin-film nanofibrous composite (TFNC) membranes for purification of wastewater (e.g., bilge water) have been prepared by using cellulose, chitin, and a cellulose-chitin blend, regenerated from an ionic liquid. The structures and properties of regenerated cellulose, chitin, and a cellulose-chitin blend were analyzed with thermogravimetric analysis (TGA) and wide-angle X-ray diffraction (WAXD). The surface morphology, pore size and pore size distribution of TFNC membranes were determined by SEM images and molecular weight cut-off (MWCO) methods. An oil/water emulsion, a model of bilge water, was used as the feed solution, and the permeation flux and rejection ratio of the membranes were investigated. TFNC membranes based on the cellulose-chitin blend exhibited 10 times higher permeation flux when compared with a commercial UF membrane (PAN10, Sepro) with a similar rejection ratio after filtration over a time period of up to 100 h, implying the practical feasibility of such membranes for UF applications.

  4. The development and use of small molecule inhibitors of glycosphingolipid metabolism for lysosomal storage diseases

    PubMed Central

    Shayman, James A.; Larsen, Scott D.

    2014-01-01

    Glycosphingolipid (GSL) storage diseases have been the focus of efforts to develop small molecule therapeutics from design, experimental proof of concept studies, and clinical trials. Two primary alternative strategies that have been pursued include pharmacological chaperones and GSL synthase inhibitors. There are theoretical advantages and disadvantages to each of these approaches. Pharmacological chaperones are specific for an individual glycoside hydrolase and for the specific mutation present, but no candidate chaperone has been demonstrated to be effective for all mutations leading to a given disorder. Synthase inhibitors target single enzymes such as glucosylceramide synthase and inhibit the formation of multiple GSLs. A glycolipid synthase inhibitor could potentially be used to treat multiple diseases, but at the risk of lowering nontargeted cellular GSLs that are important for normal health. The basis for these strategies and specific examples of compounds that have led to clinical trials is the focus of this review. PMID:24534703

  5. A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin, a polymer of N-acetyl-D-glucosamine, is found in fungal cell walls, but not in plants. Plant cells are capable of perceiving chitin fragments (chitooligosaccharides) to trigger plant defense. We identified a LysM receptor-like protein (AtLysM RLK1) that is required for the perception of chit...

  6. A LysM Receptor-like Kinase Plays a Critical Role in Chitin Signaling and Fungal Resistance in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin, a polymer of N-acetyl-D-glucosamine, is found in fungal cell walls, but not in plants. Plant cells are capable of perceiving chitin fragments (chitooligosaccharides) to trigger plant defense. We identified a LysM receptor-like protein (AtLysM RLK1) that is required for the perception of chit...

  7. First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera).

    PubMed

    Ehrlich, Hermann; Maldonado, Manuel; Spindler, Klaus-Dieter; Eckert, Carsten; Hanke, Thomas; Born, René; Goebel, Caren; Simon, Paul; Heinemann, Sascha; Worch, Hartmut

    2007-07-15

    The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals. PMID:17285638

  8. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    NASA Astrophysics Data System (ADS)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  9. Human isoprenoid synthase enzymes as therapeutic targets

    PubMed Central

    Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

    2014-01-01

    In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

  10. Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

    PubMed

    Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

    2013-04-01

    Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-?-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. PMID:23550753

  11. Chitin based polyurethanes using hydroxyl terminated polybutadiene. Part I: molecular engineering.

    PubMed

    Zia, Khalid Mahmood; Mahmood, Kashif; Zuber, Mohammad; Jamil, Tahir; Shafiq, Muhammad

    2013-08-01

    Chitin based polyurethanes (PUs) using hydroxyl terminated polybutadiene (HTPB) as soft segment were prepared and the structure of the proposed PU was confirmed using FTIR spectrometer. PU prepolymer was prepared using HTPB and toluene-2,4-diisocyanate (TDI), and the chain was extended with different proportions of 1,4-butane diol (BDO) and chitin. During the detailed FTIR study, it was observed that tri-functional character of chitin results in the formation of network structure due to crosslinking of the material, whereas bi-functional aliphatic diol based polyurethane produced linear PU. Hydrogen bonding between the hard segments was identified by the IR spectroscopy. The scanning electron microscope (SEM) analysis also confirmed the cross-linked structure. PMID:23643975

  12. Chitin based polyurethanes using hydroxyl terminated polybutadiene, Part II: morphological studies.

    PubMed

    Zia, Khalid Mahmood; Qureshi, Naureen Aziz; Mujahid, Mohammad; Mahmood, Kashif; Zuber, Mohammad

    2013-08-01

    Chitin-hydroxyl terminated polybutadiene (HTPB) based polyurethane (PU) was prepared and structure of the pre-designed PU was confirmed using FT-IR spectrometer. The FT-IR analysis confirmed that the crosslinking density increased with increasing the chitin contents in the final PU. During the detailed FT-IR study, it was observed that tri-functional character of chitin is responsible for the formation of network structure. The scanning electron microscope (SEM) analysis also confirmed the cross-linked structure of the material. The amount of carbon, nitrogen and oxygen elements obtained from EDX-SEM microanalysis also supported the results. The resistance in solubility against the solvent also confirmed the crosslinking behavior of the prepared polyurethane. PMID:23603077

  13. Transforming nanostructured chitin from crustacean waste into beneficial health products: a must for our society

    PubMed Central

    Morganti, P; Morganti, G; Morganti, A

    2011-01-01

    Chitin, obtained principally from crustacean waste, is a sugar-like polymer that is available at low cost. It has been shown to be bio- and ecocompatible, and has a very low level of toxicity. Recently, it has become possible to industrially produce pure chitin crystals, named “chitin nanofibrils” (CN) for their needle-like shape and nanostructured average size (240 × 5 × 7 nm). Due to their specific chemical and physical characteristics, CN may have a range of industrial applications, from its use in biomedical products and biomimetic cosmetics, to biotextiles and health foods. At present, world offshore disposal of this natural waste material is around 250 billion tons per year. It is an underutilized resource and has the potential to supply a wide range of useful products if suitably recycled, thus contributing to sustainable growth and a greener economy. PMID:24198491

  14. Effects of Chitin and Its Derivative Chitosan on Postharvest Decay of Fruits: A Review

    PubMed Central

    Zhang, Hongyin; Li, Renping; Liu, Weimin

    2011-01-01

    Considerable economic losses to harvested fruits are caused by postharvest fungal decay during transportation and storage, which can be significantly controlled by synthetic fungicides. However, considering public concern over pesticide residues in food and the environment, there is a need for safer alternatives for the control of postharvest decay to substitute synthetic fungicides. As the second most abundant biopolymer renewable source in nature, chitin and its derivative chitosan are widely used in controlling postharvest decay of fruits. This review aims to introduce the effect of chitin and chitosan on postharvest decay in fruits and the possible modes of action involved. We found most of the actions discussed in these researches rest on physiological mechanisms. All of the mechanisms are summarized to lay the groundwork for further studies which should focus on the molecular mechanisms of chitin and chitosan in controlling postharvest decay of fruits. PMID:21541034

  15. Glycogen synthase kinase 3: more than a namesake

    PubMed Central

    Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

    2009-01-01

    Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, ? protein and ? catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target. British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00085.x PMID:19366350

  16. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.

    PubMed Central

    Wilcox, C S; Welch, W J; Murad, F; Gross, S S; Taylor, G; Levi, R; Schmidt, H H

    1992-01-01

    Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission. Images PMID:1281548

  17. Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors.

    PubMed

    Peat, Andrew J; Boucheron, Joyce A; Dickerson, Scott H; Garrido, Dulce; Mills, Wendy; Peckham, Jennifer; Preugschat, Frank; Smalley, Terrence; Schweiker, Stephanie L; Wilson, Jayme R; Wang, Tony Y; Zhou, Huiqiang Q; Thomson, Stephen A

    2004-05-01

    A series of [1-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]arylhydrazones were discovered as novel inhibitors glycogen synthase kinase-3 (GSK-3). Based on initial modeling a detailed SAR was constructed. Modification of the interior binding aryl ring (Ar(1)) determined this to be a tight binding region with little room for modification. As predicted from the model, a large variety of modifications could be incorporated into the hydrazone aryl ring. This work led to GSK-3 inhibitors in the low nano-molar range. PMID:15080992

  18. Study of Fourier transform infrared spectra of cockroach nervous tissue and chitin

    NASA Astrophysics Data System (ADS)

    Ghadage, Vijay H.; Kulkarni, Gauri R.; Bhoraskar, Sudha V.

    2012-03-01

    Fourier Transform Infrared Spectroscopy (FTIR) is a very sensitive tool which is capable of providing strong insight on structural and functional changes in lipids and proteins induced by laser radiation. In the present work cockroach nervous tissue and chitin from tibia region are irradiated with Nd: YAG laser (?= 1064 nm, Power =150mW) via fiber optics (Numerical aperture=0.22, diameter = 8 ?). Nd: YAG laser exposure time is varied from 10 sec to 50 sec for nervous tissue and chitin. FTIR (Fourier Transform Infra Red spectra) of cockroach nervous tissue and chitin are compared before and after laser irradiation. The FTIR spectrum of non irradiated cockroach nervous tissue shows clearly the peaks due to O-H (Carboxylic acid), C=O (Amide I), C=C (Aromatic), N=0 (Nitro), C-H (Alkenes), CH (Aromatics). FTIR Spectra of non irradiated cockroach chitin clearly shows O-H (Carboxylic acid), C=O (Carbonyl stretch), C=C (Aromatic), N=O (Nitro), C-O, (anhydrides), C-H (Alkenes stretch) group. FTIR spectra of laser radiated nervous tissue from cockroach tibia and chitin shows significant changes in transmittance for O-H, C=O, C=C, C-H, N=O, C-O and C-H groups. The percentage transmittance increases for O-H, C=C group for exposure time 10sec, 40sec and 50 sec for nervous tissue. The percentage transmittance increases for O-H, C=C group for exposure time 10sec, 20sec, 30sec and 40 sec for chitin. The study shows clearly that FTIR spectroscopy of nervous tissue can reveal the interactions between infrared laser light and nervous tissue.

  19. Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin 1

    PubMed Central

    Ren, Yue-Ying; West, Charles A.

    1992-01-01

    Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched ?-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice. PMID:16668985

  20. Micro-CT Imaging of Denatured Chitin by Silver to Explore Honey Bee and Insect Pathologies

    PubMed Central

    Butzloff, Peter R.

    2011-01-01

    Background Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. Methodology/Principal Findings A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. Conclusions/Significance The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an additional tool for research on CCD. PMID:22110654

  1. Chitin Recognition via Chitotriosidase Promotes Pathologic Type-2 Helper T Cell Responses to Cryptococcal Infection

    PubMed Central

    Wiesner, Darin L.; Specht, Charles A.; Lee, Chrono K.; Smith, Kyle D.; Mukaremera, Liliane; Lee, S. Thera; Lee, Chun G.; Elias, Jack A.; Nielsen, Judith N.; Boulware, David R.; Bohjanen, Paul R.; Jenkins, Marc K.; Levitz, Stuart M.; Nielsen, Kirsten

    2015-01-01

    Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection. PMID:25764512

  2. Chitinous fibrils in the lorica of the flagellate chrysophyte Poteriochromonas stipitata (syn. Ochromonas malhamensis)

    PubMed Central

    1977-01-01

    Ordered microfibrils are formed on the membrane of the cytoplasmic tail of the alga Poteriochromonas after attachment to a substrate. The ultrastructure of native and extracted stalk fibrils was studied with electron microscope methods. In addition, the structural polysaccharide was characterized by hydrolyses, separation of the monomers by thin- layer chromatography, gas-liquid chromatography and amino acid analysis, and by X-ray diffraction. The alkali-resistant fibrils yielded mostly glucosamine upon extensive hydrolysis, and showed X-ray diffraction patterns similar to those of fugal chitin. It is concluded that the resistant core of the fibrils is chitinous. PMID:858740

  3. Isolation and identification of chitin in three-dimensional skeleton of Aplysina fistularis marine sponge.

    PubMed

    Wysokowski, Marcin; Bazhenov, Vasilii V; Tsurkan, Mikhail V; Galli, Roberta; Stelling, Allison L; Stöcker, Hartmut; Kaiser, Sabine; Niederschlag, Elke; Gärtner, Günter; Behm, Thomas; Ilan, Micha; Petrenko, Alexander Y; Jesionowski, Teofil; Ehrlich, Hermann

    2013-11-01

    The recent discovery of chitin within skeletons of numerous marine and freshwater sponges (Porifera) stimulates further experiments to identify this structural aminopolysaccharide in new species of these aquatical animals. Aplysina fistularis (Verongida: Demospongiae: Porifera) is well known to produce biologically active bromotyrosines. Here, we present a detailed study of the structural and physico-chemical properties of the three-dimensional skeletal scaffolds of this sponge. Calcofluor white staining, Raman and IR spectroscopy, ESI-MS as well as chitinase digestion test were applied in order to unequivocally prove the first discovery of ?-chitin in skeleton of A. fistularis. PMID:23994783

  4. Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source

    PubMed Central

    2013-01-01

    Background Bioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis. Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus. Results A marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis. A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme. Conclusions Wild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/?1 frameshifting. PMID:23391022

  5. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    PubMed Central

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2010-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to the extraordinary structural diversity of naturally occurring polyketides. PMID:19362634

  6. Assaying ATP Synthase Rotor Activity

    NASA Astrophysics Data System (ADS)

    Maguire, D.; Shah, J.; McCabe, M.

    With the development of laser excitation and detection systems, it has become possible to consider analyses that have otherwise been beyond the capacity of experimentalists. In this investigation, a case is made for the development of a device to analyse ATP synthase activity in-vivo. Such analyses ultimately have applications in clinical practice, particularly in the field of mitochondrial disorders, of both nuclear and mitochondrial DNA origin.

  7. Combination therapy targeting ectopic ATP synthase and 26S proteasome induces ER stress in breast cancer cells

    PubMed Central

    Chang, H-Y; Huang, T-C; Chen, N-N; Huang, H-C; Juan, H-F

    2014-01-01

    F1Fo ATP synthase is present in all organisms and is predominantly located on the inner membrane of mitochondria in eukaryotic cells. The present study demonstrated that ATP synthase and electron transport chain complexes were ectopically expressed on the surface of breast cancer cells and could serve as a potent anticancer target. We investigated the anticancer effects of the ATP synthase inhibitor citreoviridin on breast cancer cells through proteomic approaches and revealed that differentially expressed proteins in cell cycle regulation and in the unfolded protein response were functionally enriched. We showed that citreoviridin triggered PERK-mediated eIF2? phosphorylation, which in turn attenuated general protein synthesis and led to cell cycle arrest in the G0/G1 phase. We further showed that the combination of citreoviridin and the 26S proteasome inhibitor bortezomib could improve the anticancer activity by enhancing ER stress, by ameliorating citreoviridin-caused cyclin D3 compensation, and by contributing to CDK1 deactivation and PCNA downregulation. More interestingly, the combined treatment triggered lethality through unusual non-apoptotic caspase- and autophagy-independent cell death with a cytoplasmic vacuolization phenotype. The results imply that by boosting ER stress, the combination of ATP synthase inhibitor citreoviridin and 26S proteasome inhibitor bortezomib could potentially be an effective therapeutic strategy against breast cancer. PMID:25429617

  8. The Mechanistic Investigation of the Bacterial Thiamin Thiazole Synthase and the Eukaryotic Thiamin Pyrimidine Synthase 

    E-print Network

    Lai, Rung Yi

    2015-05-26

    , the Bacillus subtilis thiazole synthase (ThiG) and Candida albicans pyrimidine synthase (THI5p) were investigated. The unanticipated reverse reaction of ThiG and the thiazole tautomer generated an intermediate, which was characterized by mass spectrometry...

  9. Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase

    SciTech Connect

    Maley, F.; Maley, G.F.

    1983-01-01

    It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.

  10. Human Cystathionine-?-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium

    PubMed Central

    d’Emmanuele di Villa Bianca, Roberta; Donnarumm, Erminia; Russo, Annapina; Fusco, Ferdinando; Ianaro, Angela; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

    2015-01-01

    Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-?-synthase. Here, we demonstrate that human cystathionine-?-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-?-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-?-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-?-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-?-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-?-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity. PMID:26368121

  11. 5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

    PubMed Central

    Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

    1995-01-01

    A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

  12. Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum

    PubMed Central

    Liu, Wenting; Feng, Xinxin; Zheng, Yingying; Huang, Chun-Hsiang; Nakano, Chiaki; Hoshino, Tsutomu; Bogue, Shannon; Ko, Tzu-Ping; Chen, Chun-Chi; Cui, Yunfeng; Li, Jian; Wang, Iren; Hsu, Shang-Te Danny; Oldfield, Eric; Guo, Rey-Ting

    2014-01-01

    We report the first X-ray crystal structure of ent-kaur-16-ene synthase from Bradyrhizobium japonicum, together with the results of a site-directed mutagenesis investigation into catalytic activity. The structure is very similar to that of the ? domains of modern plant terpene cyclases, a result that is of interest since it has been proposed that many plant terpene cyclases may have arisen from bacterial diterpene cyclases. The ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues. A bisphosphonate inhibitor binds to the same site. In the kaurene synthase from the moss Physcomitrella patens, 16-?-hydroxy-ent-kaurane as well as kaurene are produced since Leu and Tyr in the P. patens kaurene synthase active site are replaced by smaller residues enabling carbocation quenching by water. Overall, the results represent the first structure determination of a bacterial diterpene cyclase, providing insights into catalytic activity, as well as structural comparisons with diverse terpene synthases and cyclases which clearly separate the terpene cyclases from other terpene synthases having highly ?-helical structures. PMID:25269599

  13. Heterologous expression of the antifungal ?-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

    PubMed

    Mehmood, Muhammad A; Latif, Mamoona; Hussain, Khadim; Gull, Munazza; Latif, Farooq; Rajoka, Muhammad I

    2015-01-01

    The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to ?-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products. PMID:25182053

  14. Suppression of Meloidogyne hapla and Its Damage to Lettuce Grown in a Mineral Soil Amended with Chitin and Biocontrol Organisms

    PubMed Central

    Chen, J.; Abawi, G. S.; Zuckerman, B. M.

    1999-01-01

    Chitin was used as soil amendment in fiberglass field microplots, alone or with one or a combination of two to three species of Hirsutella rhossiliensis, Paecilomyces marquandii, Verticillium chlamydosporium, Bacillus thuringiensis, and Streptomyces costaricanus. Sudangrass and rapeseed were planted as cover crops and incorporated into soil as green manure amendments. Chitin amendment alone increased the marketable yield of lettuce in 1995 and reduced root-galling ratings and the reproduction of Meloidogyne hapla in both 1995 and 1996. Green manure amendments of sudangrass and rapeseed increased total and marketable yields of lettuce, and decreased root-galling ratings and the reproduction of M. hapla in 1996. Hirsutella rhossiliensis in combination with chitin increased total yield of lettuce over the chitin amendment alone in 1995. The combination of B. thuringiensis, S. costaricanus, and chitin either with or without P. marquandii increased total yield of lettuce over the chitin amendment alone in 1996. In most cases, however, the nematode-antagonistic organisms did not improve lettuce yield or further suppression of M. hapla compared to the chitin amendment alone. The introduced fungi were recoverable from the infested soil. The rifampicin-resistant mutant of B. thuringiensis was not isolated at the end of the season. PMID:19270942

  15. Participation of chitin-binding peroxidase isoforms in the wilt pathogenesis of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific chitin-binding isozymes of peroxidase (POX) play an important role in pathogenesis of plant diseases caused with fungi. We studied the dynamics of peroxidase activity in two varieties of cotton (Gossypium hirsutum L.); one was a susceptible and the other resistant to the plant pathogen Vert...

  16. MOLECULAR TRACERS FOR SMOKE FROM CHARRING/BURNING OF CHITIN BIOPOLYMER. (R823990)

    EPA Science Inventory

    Abstract

    Monosaccharide derivatives from the breakdown of cellulose are the major organic components of smoke particles emitted to the atmosphere from biomass burning. In urban areas a related biopolymer, chitin, may contribute markers to smoke from grilling/charring o...

  17. First Evidence of Chitin as a Component of the Skeletal Fibers of Marine Sponges.

    E-print Network

    Maldonado, Manuel

    . Verongidae (Demospongia: Porifera) HERMANN EHRLICH1Ã, MANUEL MALDONADO2 , KLAUS-DIETER SPINDLER3 , CARSTEN, Germany ABSTRACT The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also. Part I. verongidae (demospongia: porifera). J. Exp. Zool. (Mol. Dev. Evol.) 308B:347­356. Chitin is

  18. Characterization of Organics Consistent with ?-Chitin Preserved in the Late Eocene Cuttlefish Mississaepia mississippiensis

    PubMed Central

    Weaver, Patricia G.; Doguzhaeva, Larisa A.; Lawver, Daniel R.; Tacker, R. Christopher; Ciampaglio, Charles N.; Crate, Jon M.; Zheng, Wenxia

    2011-01-01

    Background Preservation of original organic components in fossils across geological time is controversial, but the potential such molecules have for elucidating evolutionary processes and phylogenetic relationships is invaluable. Chitin is one such molecule. Ancient chitin has been recovered from both terrestrial and marine arthropods, but prior to this study had not been recovered from fossil marine mollusks. Methodology/Principal Findings Organics consistent with ?-chitin are recovered in cuttlebones of Mississaepia mississippiensis from the Late Eocene (34.36 million years ago) marine clays of Hinds County, Mississippi, USA. These organics were determined and characterized through comparisons with extant taxa using Scanning Electron Microscopy/Energy Dispersive Spectrometry (SEM/EDS), Field Emission Scanning Electron Microscopy (Hyperprobe), Fourier Transmission Infrared Spectroscopy (FTIR) and Immunohistochemistry (IHC). Conclusions/Significance Our study presents the first evidence for organics consistent with chitin from an ancient marine mollusk and discusses how these organics have been degraded over time. As mechanisms for their preservation, we propose that the inorganic/organic lamination of the cuttlebone, combined with a suboxic depositional environment with available free Fe2+ ions, inhibited microbial or enzymatic degradation. PMID:22132239

  19. Reduction of the Algicidal Properties of Copper and Mercury Ions by Chitin and Chitosan.

    ERIC Educational Resources Information Center

    Blair, Hal S.; And Others

    1982-01-01

    When chitin and chitosan were added to growing cultures of Chlorella containing various quantities of toxic metals (copper and mercury), it was found that the presence of these materials reduced the toxic effect of the metals. Background information, procedures, and results are provided for this experiment. (Author/JN)

  20. Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6 – 9 were ...

  1. A New Type I Peritrophic Membrane Protein from Larval Holotrichia oblita (Coleoptera: Melolonthidae) Binds to Chitin

    PubMed Central

    Liu, Xiaomin; Li, Jie; Guo, Wei; Li, Ruijun; Zhao, Dan; Li, Xinna

    2014-01-01

    Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13–17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation. PMID:24758927

  2. PROPERTIES OF CATALYTIC, LINKER AND CHITIN-BINDING DOMAINS OF INSECT CHITINASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in i...

  3. Sexual Dichromatism of the Damselfly Calopteryx japonica Caused by a Melanin-Chitin Multilayer in the

    E-print Network

    Sexual Dichromatism of the Damselfly Calopteryx japonica Caused by a Melanin-Chitin Multilayer causing iridescence. Citation: Stavenga DG, Leertouwer HL, Hariyama T, De Raedt HA, Wilts BD (2012) Sexual Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium

  4. Pre-irradiation grafting of acrylonitrile onto chitin for adsorption of arsenic in water

    NASA Astrophysics Data System (ADS)

    Hanh, Truong Thi; Huy, Ha Thuc; Hien, Nguyen Quoc

    2015-01-01

    Radiation-induced grafting is an effective technique for preparation of novel materials. In this study, partially deacetylated chitin with deacetylation degree (DDA) of about 40% was graft-copolymerized with acrylonitrile (AN) by a ?-ray pre-irradiation method. The maximal grafting degree of AN onto pre-irradiated chitin at 25±1.2 kGy was 114% for AN concentration in dimethylformamide of 40% (v/v) at 70 °C for 8 h. The mixture ratio of 0.1 N NH2OH·HCl to 0.1 N NaOH was selected to be 7:3 (v/v) for amidoxime conversion of cyano-groups on grafted chitin (Chi-g-AN). The characteristics of modified chitin were depicted by the FT-IR spectra, BET area and SEM images. Adsorption equilibrium of As(III) onto Chi-g-AN converted amidoxime (Chi-g-AN-C) fits with the Langmuir model and the maximal adsorption capacity was 19.724 mg/g. The break-through times of As(III) on Chi-g-AN-C in column adsorption experiments increased with the increase in bed depths.

  5. Draft Genome Sequence of Amantichitinum ursilacus IGB-41, a New Chitin-Degrading Bacterium

    PubMed Central

    Kirstahler, Philipp; Günther, Michael; Grumaz, Christian; Lindemann, Elena; Rupp, Steffen; Zibek, Susanne

    2015-01-01

    Amantichitinum ursilacus IGB-41 is a new species of chitin-degrading bacterium isolated from soil, which secretes potential industrial enzymes. The genome of A. ursilacus was sequenced, and the gene set encoding chitinases was identified. Here, we present the draft genome of 4.9 Mb, comprising 38 contigs, and the corresponding annotation. PMID:26586877

  6. Refractive index and dispersion of butterfly chitin and bird keratin measured by polarizing

    E-print Network

    Refractive index and dispersion of butterfly chitin and bird keratin measured by polarizing the wavelength dependence of the refractive index of butterfly wing scales and bird feathers. The refractive index values of the glass scales of the butterfly Graphium sarpedon are, at wavelengths 400, 500 and 600

  7. Improving sensitivity of direct microscopy for detection of acid-fast bacilli in sputum: use of chitin in mucus digestion.

    PubMed

    Farnia, P; Mohammadi, F; Zarifi, Z; Tabatabee, D J; Ganavi, J; Ghazisaeedi, K; Farnia, P K; Gheydi, M; Bahadori, M; Masjedi, M R; Velayati, A A

    2002-02-01

    In order to try to improve the results of direct smear microscopy, we used the mucus-digesting quality of chitin in tuberculosis (TB) laboratories. For this purpose, a total of 430 sputum specimens were processed by the N-acetyl-L-cysteine concentration, sodium hypochlorite (NaOCl) liquefaction, chitin sedimentation, and direct microscopy methods. Then, the smear sensitivity for acid-fast bacillus detection by chitin-treated sputum was compared with the sensitivity of smears prepared by other methods. Our results showed that the chitin solution took less time to completely homogenize the mucoid sputum than did the N-acetyl-L-cysteine and NaOCl methods. The N-acetyl-L-cysteine concentration method demonstrated sensitivity and specificity levels of 83 and 97%, respectively. In comparison, the sensitivity of chitin sedimentation was 80%, with a specificity of 96.7%. The NaOCl liquefaction method showed a sensitivity of 78%, with a specificity of 96%. Finally, the sensitivity of direct microscopy was lower than those of the other tested methods and was only 46%, with a specificity of 90%. The chitin and NaOCl liquefaction methods are both easy to perform, and they do not require additional equipment (centrifuges). Also, our results demonstrated that the chitin method is less time-consuming than the NaOCl method, since only 30 min of incubation is required to bring complete sedimentation of bacilli in chitin-treated sputum whereas the NaOCl method needs 10 to 12 h to give the same results in the same sputum specimens. Therefore, the chitin liquefaction and sedimentation method may provide better results in TB laboratories of developing countries than the N-acetyl-L-cysteine concentration, NaOCl overnight sedimentation, and direct smear microscopy methods. PMID:11825964

  8. CFTR Inhibitors

    PubMed Central

    Verkman, Alan S.; Synder, David; Tradtrantip, Lukmanee; Thiagarajah, Jay R.; Anderson, Marc O.

    2014-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl? channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. PMID:23331030

  9. GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS

    PubMed Central

    Eldar-Finkelman, Hagit; Martinez, Ana

    2011-01-01

    Inhibiting glycogen synthase kinase-3 (GSK-3) activity via pharmacological intervention has become an important strategy for treating neurodegenerative and psychiatric disorders. The known GSK-3 inhibitors are of diverse chemotypes and mechanisms of action and include compounds isolated from natural sources, cations, synthetic small-molecule ATP-competitive inhibitors, non-ATP-competitive inhibitors, and substrate–competitive inhibitors. Here we describe the variety of GSK-3 inhibitors with a specific emphasis on their biological activities in neurons and neurological disorders. We further highlight our current progress in the development of non-ATP-competitive inhibitors of GSK-3. The available data raise the hope that one or more of these drug design approaches will prove successful at stabilizing or even reversing the aberrant neuropathology and cognitive deficits of certain central nervous system disorders. PMID:22065134

  10. Electrochemical study of functionalization on the surface of a chitin/platinum-modified glassy carbon paste electrode.

    PubMed

    Sugawara, Kazuharu; Yugami, Asako; Terui, Norifumi; Kuramitz, Hideki

    2009-11-01

    To functionalize chitin surfaces using proteins, we developed a glucose oxidase (GOD)-chitin/platinum-modified glassy carbon paste electrode (GCPE) as a model. In a weakly acidic solution, negatively charged GOD were immobilized by the protonated acetylamide groups on chitin. When the electrode was immersed in a solution containing GOD, the enzyme was readily immobilized due to the electrostatic interaction. In addition, measurements were performed using electrodes made with powders of different sizes because sensor performance depends on the particle sizes of glassy carbon powder. PMID:19907096

  11. Deoxyribonuclease inhibitors.

    PubMed

    Kolarevic, Ana; Yancheva, Denitsa; Kocic, Gordana; Smelcerovic, Andrija

    2014-12-17

    Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or modify their activities. This review focuses on DNase inhibitors. Some DNase inhibitors have been isolated from various natural sources, such as humans, animals (beef, calf, rabbit and rat), plants (Nicotiana tabacum), and microorganisms (some Streptomyces and Adenovirus species, Micromonospora echinospora and Escherichia coli), while others have been obtained by chemical synthesis. They differ in chemical structure (various proteins, nucleotides, anthracycline and aminoglycoside antibiotics, synthetic organic and inorganic compounds) and mechanism of action (forming complexes with DNases or DNA). Some of the inhibitors are specific toward only one type of DNase, while others are active towards two or more. Physico-chemical properties of DNase inhibitors are calculated using the Molinspiration tool and most of them meet all criteria for good solubility and permeability. DNase inhibitors may be used as pharmaceuticals for preventing, monitoring and treating various diseases. PMID:25042005

  12. New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

    SciTech Connect

    Durner, J.; Gailus, V.; Boeger, P. )

    1991-04-01

    The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyrvuate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, and initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of ({sup 14}C)chlorsulfuron and ({sup 14}C)imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency.

  13. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  14. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline

    PubMed Central

    Preiss, Laura; Langer, Julian D.; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E. G.; Koul, Anil; Meier, Thomas

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring’s ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens. PMID:26601184

  15. Fatty acid synthase as a potential therapeutic target in cancer

    PubMed Central

    Flavin, Richard; Peluso, Stephane; Nguyen, Paul L; Loda, Massimo

    2011-01-01

    Fatty acid synthase (FASN) is a key enzyme involved in neoplastic lipogenesis. Overexpression of FASN is common in many cancers, and accumulating evidence suggests that it is a metabolic oncogene with an important role in tumor growth and survival, making it an attractive target for cancer therapy. Early small-molecule FASN inhibitors such as cerulenin, C75 and orlistat have been shown to induce apoptosis in several cancer cell lines and to induce tumor growth delay in several cancer xenograft models but their mechanism is still not well understood. These molecules suffer from pharmacological limitations and weight loss as a side effect that prevent their development as systemic drugs. Several potent inhibitors have recently been reported that may help to unravel and exploit the full potential of FASN as a target for cancer therapy in the near future. Furthermore, novel sources of FASN inhibitors, such as green tea and dietary soy, make both dietary manipulation and chemoprevention potential alternative modes of therapy in the future. PMID:20373869

  16. Squalene Synthase As a Target for Chagas Disease Therapeutics

    PubMed Central

    Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A.; Wang, Andrew H.-J.; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

    2014-01-01

    Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. PMID:24789335

  17. Rhizobium tropici chromosomal citrate synthase gene.

    PubMed Central

    Hernández-Lucas, I; Pardo, M A; Segovia, L; Miranda, J; Martínez-Romero, E

    1995-01-01

    Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici. PMID:8526514

  18. Mitochondrial F0F1-ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone

    PubMed Central

    Cumero, S; Fogolari, F; Domenis, R; Zucchi, R; Mavelli, I; Contessi, S

    2012-01-01

    BACKGROUND AND PURPOSE 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F0F1-ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH Kinetic analyses were performed on F0F1-ATP synthase in sub-mitochondrial particles and soluble F1-ATPase. Activity assays and immunodetection of the inhibitor protein IF1 were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS T1AM was a non-competitive inhibitor of F0F1-ATP synthase whose binding was mutually exclusive with that of the inhibitors IF1 and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF1 binding site, causing IF1 release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F0F1-ATP synthase consistent with displacement of endogenous IF1,, reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS Effects of T1AM on F0F1-ATP synthase were twofold: IF1 displacement and enzyme inhibition. By targeting F0F1-ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF1 and aurovertin binding sites are described. PMID:22452346

  19. Chitin and Chitosan as Multipurpose Natural Polymers for Groundwater Arsenic Removal and As2O3 Delivery in Tumor Therapy

    PubMed Central

    Da Sacco, Letizia; Masotti, Andrea

    2010-01-01

    Chitin and chitosan are natural polysaccharide polymers. These polymers have been used in several agricultural, food protection and nutraceutical applications. Moreover, chitin and chitosan have been also used in biomedical and biotechnological applications as drug delivery systems or in pharmaceutical formulations. So far, there are only few studies dealing with arsenic (As) removal from groundwater using chitin or chitosan and no evidence of the use of these natural polymers for arsenic trioxide (As2O3) delivery in tumor therapy. Here we suggest that chitin and/or chitosan might have the right properties to be employed as efficient polymers for such applications. Besides, nanotechnology offers suitable tools for the fabrication of novel nanostructured materials of natural origin. Since different nanostructured materials have already been employed successfully in various multidisciplinary fields, we expect that the integration of nanotechnology and natural polymer chemistry will further lead to innovative applications for environment and medicine. PMID:20559486

  20. Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

    PubMed

    Bajaj, Mini; Freiberg, Andrea; Winter, Josef; Xu, Youmei; Gallert, Claudia

    2015-11-01

    Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan. PMID:26227412

  1. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

    NASA Astrophysics Data System (ADS)

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarnés, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-03-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

  2. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  3. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  4. Marine hydroid perisarc: a chitin- and melanin-reinforced composite with DOPA-iron(III) complexes.

    PubMed

    Hwang, Dong Soo; Masic, Admir; Prajatelistia, Ekavianty; Iordachescu, Mihaela; Waite, J Herbert

    2013-09-01

    Many marine invertebrates utilize biomacromolecules as building blocks to form their load-bearing tissues. These polymeric tissues are appealing for their unusual physical and mechanical properties, including high hardness and stiffness, toughness and low density. Here, a marine hydroid perisarc of Aglaophenia latirostris was investigated to understand how nature designs a stiff, tough and lightweight sheathing structure. Chitin, protein and a melanin-like pigment, were found to represent 10, 17 and 60 wt.% of the perisarc, respectively. Interestingly, similar to the adhesive and coating of marine mussel byssus, a DOPA (3,4-dihydroxyphenylalanine) containing protein and iron were detected in the perisarc. Resonance Raman microprobe analysis of perisarc indicates the presence of catechol-iron(III) complexes in situ, but it remains to be determined whether the DOPA-iron(III) interaction plays a cohesive role in holding the protein, chitin and melanin networks together. PMID:23791678

  5. Pyrolysis of chitin biomass: TG-MS analysis and solid char residue characterization.

    PubMed

    Qiao, Yan; Chen, Shuai; Liu, Ying; Sun, Haizhen; Jia, Shiyu; Shi, Junyan; Pedersen, Christian Marcus; Wang, Yingxiong; Hou, Xianglin

    2015-11-20

    The thermal degradation of chitin biomass with various molecular structures was investigated by thermogravimetric analysis (TG), and the gaseous products were analyzed by connected mass spectroscopy (MS). The chemical structure and morphology of char residues collected at 750°C using the model substrates GlcNH2 and GlcNAc, were characterized systematically. The experimental results disclosed that one main mass loss stage was observed for each substrate. Chitosan samples with high molecular weight shown the more thermal stability, and chitin showed the highest thermal stability. Additionally, it was found that catalysts play a significant role during the pyrolysis. The gaseous evolution components, including NH3, H2O, CO, and CO2 were observed by on line MS. The experimental results disclosed that the obtained carbonaceous materials had lost the original hydrocarbon structure totally, and transformed into an aromatic structure with high carbon and nitrogen content, which was identified by XPS and solid state NMR. PMID:26344268

  6. Synthesis of carboxymethyl chitin in aqueous solution and its thermo- and pH-sensitive behaviors.

    PubMed

    Liu, Hui; Yang, Qizhi; Zhang, Lina; Zhuo, Renxi; Jiang, Xulin

    2016-02-10

    Homogenous modification of natural chitin offers the advantage of fair structure control. In this work, novel carboxymethyl chitins (CMCHs) with broad range of degree of substitution (DS: 0.035 to 0.74), high degree of acetylation (DA) and little de-polymerization were synthesized homogeneously in aqueous NaOH/urea solution. The simultaneous determination of DA, DS and carboxymethylation fraction at C3 and C6 for these CMCHs was achieved by proton NMR in acidic deuterated aqueous solution for the first time. Due to the good homogeneity, the prepared CMCH-4 with lower DS of carboxymethylation exhibited, for the first time to our knowledge, dual thermo- and pH-sensitive properties. The nontoxic thermo-sensitive polymer systems gel at body temperature (37°C) in physiological condition, which is very useful as injectable hydrogels for drug delivery and tissue engineering. PMID:26686169

  7. Water in Crystalline Fibers of Dihydrate ?-Chitin Results in Unexpected Absence of Intramolecular Hydrogen Bonding

    PubMed Central

    Sawada, Daisuke; Nishiyama, Yoshiharu; Langan, Paul; Forsyth, V. Trevor; Kimura, Satoshi; Wada, Masahisa

    2012-01-01

    The complete crystal structure (including hydrogen) of dihydrate ?-chitin, a homopolymer of N-acetylglucosamine hydrate, was determined using high-resolution X-ray and neutron fiber diffraction data collected from bathophilous tubeworm Lamellibrachia satsuma. Two water molecules per N-acetylglucosamine residue are clearly localized in the structure and these participate in most of the hydrogen bonds. The conformation of the labile acetamide groups and hydroxymethyl groups are similar to those found in anhydrous ?-chitin, but more relaxed. Unexpectedly, the intrachain O3-H…O5 hydrogen bond typically observed for crystalline ?,1–4 glycans is absent, providing important insights into its relative importance and its relationship to solvation. PMID:22724007

  8. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  9. Anticancer and Anti-Inflammatory Properties of Chitin and Chitosan Oligosaccharides

    PubMed Central

    Azuma, Kazuo; Osaki, Tomohiro; Minami, Saburo; Okamoto, Yoshiharu

    2015-01-01

    Previous reports indicate that N-acetyl-d-glucosamine oligomers (chitin oligosaccharide; NACOS) and d-glucosamine oligomers (chitosan oligosaccharide; COS) have various biological activities, especially against cancer and inflammation. In this review, we have summarized the findings of previous investigations that have focused on anticancer or anti-inflammatory properties of NACOS and COS. Moreover, we have introduced recent evaluation of NACOS and COS as functional foods against cancer and inflammatory disease. PMID:25594943

  10. Studies on the Chitin Binding Property of Novel Cysteine-Rich Peptides from Alternanthera sessilis.

    PubMed

    Kini, Shruthi G; Nguyen, Phuong Q T; Weissbach, Sophie; Mallagaray, Alvaro; Shin, Joon; Yoon, Ho Sup; Tam, James P

    2015-11-01

    Hevein-like peptides make up a family of cysteine-rich peptides (CRPs) and play a role in plants in their defense against insects and fungal pathogens. In this study, we report the isolation and characterization of six hevein-like peptides, aSG1-G3 and aSR1-R3, collectively named altides from green and red varieties of Alternanthera sessilis, a perennial herb belonging to the Amaranthaceae family. Proteomic analysis of altides revealed they contain six cysteines (6C), seven glycines, four prolines, and a conserved chitin-binding domain (SXYGY/SXFGY). Thus far, only four 6C-hevein-like peptides have been isolated and characterized; hence, our study expands the existing library of these peptides. Nuclear magnetic resonance (NMR) study of altides showed its three disulfide bonds were arranged in a cystine knot motif. As a consequence of this disulfide arrangement, they are stable against thermal and enzymatic degradation. Gene cloning studies revealed altides contain a three-domain precursor with an endoplasmic reticulum signal peptide followed by a mature CRP domain and a short C-terminal tail. This indicates that the biosynthesis of altides is through the secretory pathway. (1)H NMR titration experiments showed that the 29-30-amino acid altides bind to chitin oligomers with dissociation constants in the micromolar range. Aromatic residues in the chitin-binding domain of altides were involved in the binding interaction. To the best of our knowledge, aSR1 is the smallest hevein-like peptide with a dissociation constant toward chitotriose comparable to those of hevein and other hevein-like peptides. Together, our study expands the existing library of 6C-hevein-like peptides and provides insights into their structure, biosynthesis, and interaction with chitin oligosaccharides. PMID:26467613

  11. New photocatalyst based on graphene oxide/chitin for degradation of dyes under sunlight.

    PubMed

    Wang, Yuntao; Pei, Yaqiong; Xiong, Wenfei; Liu, Tingguo; Li, Jing; Liu, Shilin; Li, Bin

    2015-11-01

    Sunlight photocatalyst was fabricated by in situ synthesis of Cu2O in the regenerated chitin (RC)/graphene oxide (GO) composite film, where the porous chitin film was used as the microreactor for the formation of nano Cu2O. Nano Cu2O was immobilized and evenly distributed in the matrix and Cu2O tended to grow on the GO sheets. Cu2O inside the matrix excite and generate free photoelectrons and electron holes, which was responsible for the degradation of dyes, while GO transferred the yielded photoelectrons to prevent the generation of local high potential zone and induce the chain degradation at more points. So it was found that the porous chitin film could load Cu2O and graphene at the same time, controlling the size of Cu2O and leading to easy recycle and reuse of the photocatalyst. Moreover, the introduction of GO has dramatically improved the photocatalytic activity of Cu2O in the Cu2O/GO/RC film, showing great potential application in wastewater treatment utilizing solar energy. PMID:26299711

  12. Chitin and Chitosan Preparation from Marine Sources. Structure, Properties and Applications

    PubMed Central

    Younes, Islem; Rinaudo, Marguerite

    2015-01-01

    This review describes the most common methods for recovery of chitin from marine organisms. In depth, both enzymatic and chemical treatments for the step of deproteinization are compared, as well as different conditions for demineralization. The conditions of chitosan preparation are also discussed, since they significantly impact the synthesis of chitosan with varying degree of acetylation (DA) and molecular weight (MW). In addition, the main characterization techniques applied for chitin and chitosan are recalled, pointing out the role of their solubility in relation with the chemical structure (mainly the acetyl group distribution along the backbone). Biological activities are also presented, such as: antibacterial, antifungal, antitumor and antioxidant. Interestingly, the relationship between chemical structure and biological activity is demonstrated for chitosan molecules with different DA and MW and homogeneous distribution of acetyl groups for the first time. In the end, several selected pharmaceutical and biomedical applications are presented, in which chitin and chitosan are recognized as new biomaterials taking advantage of their biocompatibility and biodegradability. PMID:25738328

  13. Bio-responsive chitin-poly(L-lactic acid) composite nanogels for liver cancer.

    PubMed

    Arunraj, T R; Sanoj Rejinold, N; Ashwin Kumar, N; Jayakumar, R

    2014-01-01

    Hepatic carcinoma (HCC) is one of the most common cancer and its treatment has been considered a therapeutic challenge. Doxorubicin (Dox) is one of the most important chemotherapeutic agents used in the treatment for liver cancer. However, the efficacy of Dox therapy is restricted by the dose-dependent toxic side effects. To overcome the cardiotoxicity of Dox as well as the current problems of conventional modality treatment of HCC, we developed a locally injectable, biodegradable, and pH sensitive composite nanogels for site specific delivery. Both control and Dox loaded composite nanogel systems were analyzed by DLS, SEM, FTIR and TG/DTA. The size ranges of the control composite nanogels and their drug loaded counterparts were found to be 90±20 and 270±20 nm, respectively. The control chitin-PLA CNGs and Dox-chitin-PLA CNGs showed higher swelling and degradation in acidic pH. Drug entrapment efficiency and in vitro drug release studies were carried out and showed a higher drug release at acidic pH compared to neutral pH. Cellular internalization of the nanogel systems was confirmed by fluorescent microscopy. The cytotoxicity of the composite nanogels was analyzed toward HepG2 (human liver cancer) cell lines. Furthermore, the results of in vitro hemolytic assay and coagulation assay substantiate the blood compatibility of the system. Overall Dox-chitin-PLA CNGs system could be a promising anticancer drug delivery system for liver cancer therapy. PMID:24129331

  14. Binary gene expression patterning of the molt cycle: the case of chitin metabolism.

    PubMed

    Abehsera, Shai; Glazer, Lilah; Tynyakov, Jenny; Plaschkes, Inbar; Chalifa-Caspi, Vered; Khalaila, Isam; Aflalo, Eliahu D; Sagi, Amir

    2014-01-01

    In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish Cherax quadricarinatus, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes. PMID:25919476

  15. Fabrication and feasibility study of an absorbable diacetyl chitin surgical suture for wound healing.

    PubMed

    Shao, Kai; Han, Baoqin; Gao, Jinning; Jiang, Zhiwen; Liu, Weizhi; Liu, Wanshun; Liang, Ye

    2016-01-01

    Diacetyl chitin (DAC) is an acidylated chitin obtained using acetic anhydride mixed perchloric acid system. By wet spinning and weaving technique, DAC has been successfully developed into a novel absorbable surgical suture. Thanks to the unique properties of chitins, the potential application of this novel monocomponent multifilament DAC suture may break the monopoly of synthetic polymer sutures in wound closure area. In this study, DAC was synthesized and characterized by multiple approaches including elemental analysis, Fourier transform infrared spectrometry (FTIR), and X-ray diffraction (XRD). In addition, we performed the feasibility assessment of DAC suture (USP 2-0) as absorbable suture for wound healing. Several lines of evidences suggested that DAC suture had comparable mechanical properties as synthetic polymer sutures. Moreover, DAC suture retained approximately 63% of the original strength at 14 days and completely absorbed in 42 days with no remarkable tissue reaction in vivo. Most important of all, DAC suture significantly promoted skin regeneration with faster tissue reconstruction and higher wound breaking strength on a linear incisional wound model. All these results demonstrated the potential use of DAC suture in short- or middle-term wound healing, such as epithelial and connective tissue. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016:104B:116-125. PMID:25677094

  16. Co-Processed Chitin-Mannitol as a New Excipient for Oro-Dispersible Tablets

    PubMed Central

    Daraghmeh, Nidal; Chowdhry, Babur Z.; Leharne, Stephen A.; Al Omari, Mahmoud M. H.; Badwan, Adnan A.

    2015-01-01

    This study describes the preparation, characterization and performance of a novel excipient for use in oro-dispersible tablets (ODT). The excipient (Cop–CM) consists of chitin and mannitol. The excipient with optimal physicochemical properties was obtained at a chitin: mannitol ratio of 2:8 (w/w) and produced by roll compaction (RC). Differential scanning calorimetry (DSC), Fourier transform-Infrared (FT-IR), X-ray powder diffraction (XRPD) and scanning electron microscope (SEM) techniques were used to characterize Cop–CM, in addition to characterization of its powder and ODT dosage form. The effect of particle size distribution of Cop–CM was investigated and found to have no significant influence on the overall tablet physical properties. The compressibility parameter (a) for Cop–CM was calculated from a Kawakita plot and found to be higher (0.661) than that of mannitol (0.576) due to the presence of the highly compressible chitin (0.818). Montelukast sodium and domperidone ODTs produced, using Cop–CM, displayed excellent physicochemical properties. The exceptional binding, fast wetting and superdisintegration properties of Cop–CM, in comparison with commercially available co-processed ODT excipients, results in a unique multifunctional base which can successfully be used in the formulation of oro-dispersible and fast immediate release tablets. PMID:25830680

  17. Removal of 2,4-D from aqueous solutions by adsorption processes using two biopolymers: chitin and chitosan and their optical properties

    NASA Astrophysics Data System (ADS)

    El Harmoudi, H.; El Gaini, L.; Daoudi, E.; Rhazi, M.; Boughaleb, Y.; El Mhammedi, M. A.; Migalska-Zalas, A.; Bakasse, M.

    2014-07-01

    Chitin, extracted primarily from exoskeleton shellfish, is one of the most abundant biopolymer in the world. It is estimated to be produced annually almost as much as cellulose. The development of fishing activities in Morocco (coastline of 3500 km on both the Atlantic and the Mediterranean) induced an important fish industry waste. The aim of this work is to recover chitin, extracted from shellfish waste, and chitosan, produced by deacetylation of chitin, in the adsorption of organic pesticide 2,4-dichlorophenoxyacetate (2,4-D). The chitosan samples were prepared from chitin from crustacean shells. The chitin deacetylation was carried out in aqueous sodium hydroxide. Characterization of chitin and chitosan was performed by infrared spectroscopy, nuclear magnetic resonance and theoretical calculation of UV-Vis spectra and optical parameters. Analysis of theoretical spectra shows a substantial sensitivity to the hydroxyl and amino groups. The presented hydroxyl group in chitin determines the conformation of the molecule and also the stereochemistry of chemical reactions and kinetics. The presence of functional groups causes the substantial changes of both absorption spectra as well as permanent dipole moment. The adsorption of 2,4-D by chitin and chitosan was performed at different pH values, the pesticide-adsorbent contact time and initial pesticide concentration. The adsorption is described by Langmuir-type isotherm and Freundlich-type isotherm. Recovery tests of 2,4-D confirm the initial interest in using chitin and chitosan as a depolluting agent.

  18. Mechanistic Insight with HBCH2CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

    PubMed Central

    2015-01-01

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 ?M, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [3H]-saturated trimer-CoA ([3H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [3H]-sTCoA ([3H]-sT-CH2-CoA), saturated dimer-([3H]-sD-CO2H), and trimer-acid ([3H]-sT-CO2H), distinct from the expected methylene analogue of [3H]-saturated tetramer-CoA ([3H]-sTet-CH2-CoA). Detection of [3H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation. PMID:24896226

  19. Primary structure of a cerulenin-binding. beta. -ketoacyl-(acyl carrier protein) synthase from barley chloroplasts

    SciTech Connect

    Siggaard-Andersen, M.; Kauppinen, S. ); von Wettstein-Knowles, P. Univ. of Copenhagen )

    1991-05-15

    The radioactively labeled {beta}-ketoacyl thioester synthase inhibitor ({sup 3}H)cerulenin was used to tag three dimeric barley chloroplast proteins ({alpha}{alpha}, {alpha}{beta}, and {beta}{beta}) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the {beta} subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the {beta}-ketoacyl-(acyl carrier protein) (ACP) synthase I (3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41) of Escherichia coli. Under analogous experimental conditions ({sup 3}H)cerulenin tagged a single dimeric protein from spinach chloroplasts.

  20. Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Holden, Jeffrey K; Kang, Soosung; Beasley, Federico C; Cinelli, Maris A; Li, Huiying; Roy, Saurabh G; Dejam, Dillon; Edinger, Aimee L; Nizet, Victor; Silverman, Richard B; Poulos, Thomas L

    2015-06-18

    Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme. PMID:26091171

  1. The Structure of the L-myo-inositol-1-phosphate Synthase-NAD[superscript +]-2-deoxy-D-glucitol 6-(E)-Vinylhomophosphonate Complex Demands a Revision of the Enzyme Mechanism

    SciTech Connect

    Jin, Xiangshu; Foley, Kathleen M.; Geiger, James H.

    2010-11-16

    1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of D-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD{sup +} and a high-affinity inhibitor, 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-D-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD{sup +}-2-deoxy-D-glucitol 6-phosphate. There are several other conformational changes in NAD{sup +} and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.

  2. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    PubMed

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed. PMID:26123522

  3. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil ?-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the ? shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  4. Defining the Potassium Binding Region in an Apple Terpene Synthase*S?

    PubMed Central

    Green, Sol; Squire, Christopher J.; Nieuwenhuizen, Niels J.; Baker, Edward N.; Laing, William

    2009-01-01

    Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple ?-farnesene synthase (MdAFS1), which has a dependence upon potassium (K+), was used to identify active site regions that may be important for M+ binding. Protein homology modeling revealed a surface-exposed loop (H-?l loop) in MdAFS1 that fulfilled the necessary requirements for a K+ binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K+ response and strongly implicated specific residues in direct K+ binding. The role of the H-?l loop in terpene synthase K+ coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M+ binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K+ activation in other terpene synthases and establish a new role for the H-?l loop region in terpene synthase catalysis. PMID:19181671

  5. Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

    E-print Network

    Suh, Dae-Yeon

    Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli Toshio STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two that was overexpressed in Escherichia coli, we have detected bisnoryangonin (BNY, the derailed lactone after two

  6. Identification of cellulose synthase(s) in higher plants: sequence analysis of

    E-print Network

    Brown Jr., R. Malcolm

    Identification of cellulose synthase(s) in higher plants: sequence analysis of processive plants (Arabidopsis thaliana, Brassica campestris, and Oryza sativa) that exhibit a limited sequence in the processive -glycosyltransferases. Complete sequencing of an EST clone (T88271) from A. thaliana led

  7. Effect of sub- and supercritical water treatments on the physicochemical properties of crab shell chitin and its enzymatic degradation.

    PubMed

    Osada, Mitsumasa; Miura, Chika; Nakagawa, Yuko S; Kaihara, Mikio; Nikaido, Mitsuru; Totani, Kazuhide

    2015-12-10

    This study examined the effects of sub- and supercritical water pretreatments on the physicochemical properties of crab shell ?-chitin and its enzymatic degradation to obtain N,N'-diacetylchitobiose (GlcNAc)2. Following sub- and supercritical water pretreatments, the protein in the crab shell was removed and the residue of crab shell contained ?-chitin and CaCO3. Prolonged pretreatment led to ?-chitin decomposition. The reaction of pure ?-chitin in sub- and supercritical water pretreatments was investigated separately; we observed lower mean molecular weight and weaker hydrogen bonds compared with untreated ?-chitin. (GlcNAc)2 yields from enzymatic degradation of subcritical (350 °C, 7 min) and supercritical water (400 °C, 2.5 min) pretreated crab shell were 8% and 6%, compared with 0% without any pretreatment. This study shows that sub- and supercritical water pretreatments of crab shell provide to an alternative method to the use of acid and base for decalcification and deproteinization of crab shell required for (GlcNAc)2 production. PMID:26428177

  8. Resistance to AHAS inhibitor herbicides: current understanding.

    PubMed

    Yu, Qin; Powles, Stephen B

    2014-09-01

    Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined. PMID:24338926

  9. Crosstalk between Phosphodiesterase 7 and Glycogen Synthase Kinase-3: Two Relevant Therapeutic Targets for Neurological Disorders

    PubMed Central

    2014-01-01

    Chronic neuroinflammation has been increasingly recognized as a primary mechanism underlying acute brain injury and neurodegenerative diseases. Enhanced expression of diverse pro-inflammatory agents in glial cells has been shown to contribute to the cell death that takes place in these disorders. Previous data from our group have shown that different inhibitors of the cyclic adenosine monophosphate (cAMP) specific phosphodiesterase 7 (PDE7) and glycogen synthase kinase-3 (GSK-3) enzymes are potent anti-inflammatory agents in different models of brain injury. In this study, we investigated cross-talk between PDE7 and GSK-3, two relevant therapeutic targets for neurological disorders, using a chemical approach. To this end, we compared specific inhibitors of GSK-3 and PDE7 with dual inhibitors of both enzymes with regard to anti-inflammatory effects in primary cultures of glial cells treated with lipopolysaccharide. Our results show that the GSK-3 inhibitors act exclusively by inhibition of this enzyme. By contrast, PDE7 inhibitors exert their effects via inhibition of PDE7 to increase intracellular cAMP levels but also through indirect inhibition of GSK-3. Activation of protein kinase A by cAMP results in phosphorylation of Ser9 of GSK-3 and subsequent inhibition. Our results indicate that the indirect inhibition of GSK-3 by PDE7 inhibitors is an important mechanism that should be considered in the future development of pharmacological treatments. PMID:24437940

  10. Terpene synthases are widely distributed in bacteria

    PubMed Central

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  11. Terpene synthases are widely distributed in bacteria.

    PubMed

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-Ya, Kazuo; Omura, Satoshi; Cane, David E; Ikeda, Haruo

    2015-01-20

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  12. Synthesis and evaluation of novel heterocyclic inhibitors of GSK-3.

    PubMed

    Smalley, Terrence L; Peat, Andrew J; Boucheron, Joyce A; Dickerson, Scott; Garrido, Dulce; Preugschat, Frank; Schweiker, Stephanie L; Thomson, Stephen A; Wang, Tony Y

    2006-04-15

    A set of novel heterocyclic pyrimidyl hydrazones has been synthesized as inhibitors of glycogen synthase kinase-3 (GSK-3) with the most active exhibiting low nanomolar activity. Quantum mechanical calculations indicate that of the conformational factors that could determine binding affinity, the planarity of the phenyl ring in relation to the central core and the conformation of the hydrazone chain may be the most influential. PMID:16460937

  13. Effect of CaCO3/HCl pretreatment on the surface modification of chitin gel beads via graft copolymerization of 2-hydroxy ethyl methacrylate and 4-vinylpyridine.

    PubMed

    Yalinca, Zulal; Mohammed, Dana Ali Kader; Hadi, Jihad M; Yilmaz, Elvan

    2016-01-01

    Although chitin, poly(N-acetylglucosamine), possesses considerable potential as a biomaterial, it has not been as thoroughly studied as its derivative chitosan. In this study, the potential of chitin gel beads has been evaluated for surface modification via vinyl polymer grafting. Grafting behavior of two well-established vinyl monomers, namely 2-hydroxyethylmethacrylate (HEMA) and 4-vinylpyridine (4-VP) were investigated using cerium (IV) ammonium nitrate as the redox initiator with the aim of obtaining chemically functionalized more hydrophilic chitin surfaces. The intractable nature of chitin, which is one of its primary drawbacks as a grafting substrate was overcome by applying a CaCO3 treatment during bead preparation. The maximum grafting percentage of poly(HEMA) onto chitin bead without CaCO3 treatment was found to be 65%, while the value for CaCO3 treated chitin beads was 515%. The maximum grafting yield of poly(4-VP) on to CaCO3 treated chitin powder was 380% at optimum conditions. The grafting system was extensively characterized before and after grafting by FT-IR, SEM, C-13 NMR and XRD analyses. Significant improvement on the swelling capacities of chitin based gel beads in aqueous acidic, basic and neutral media was obtained. An account of the pros and cons of the system has been presented. PMID:26500177

  14. Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A bioinformatics investigation of four insect species with annotated genome sequences identified a family of genes encoding chitin deacetylase (CDA)-like proteins, with 5-9 members depending on the species. CDAs (EC 3.5.1.41) are chitin-modifying enzymes that deacetylate the b-1,4-linked N-acetylgl...

  15. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    SciTech Connect

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  16. Properties of phosphorylated thymidylate synthase.

    PubMed

    Fr?czyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Pawe?; Rogowska-Wrzesinska, Adelina; Cie?la, Joanna; Zieli?ski, Zbigniew; Nizio?, Joanna; Jarmu?a, Adam; Maj, Piotr; Go?os, Barbara; Wi?ska, Patrycja; Ostafil, Sylwia; Wa?ajtys-Rode, El?bieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  17. Chemical characteristics and cytocompatibility of collagen-based scaffold reinforced by chitin fibers for bone tissue engineering.

    PubMed

    Li, Xiaoming; Feng, Qingling; Wang, Wenjie; Cui, Fuzhai

    2006-05-01

    Chitin is a kind of seemly material to match PLLA for a scaffold, which may create an appropriate environment for the regeneration of tissues. In this study, we prepared and evaluated a new nano-hydroxyapatite/collagen/PLLA (nHACP) scaffold reinforced by chitin fibers for bone-tissue engineering. The chitin fibers were crosslinked with PLLA by dicyclohexylcarbodimide (DCC). The chemical characteristics were evaluated by Fourier transformed infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The mechanical strength was measured by compressive tests. The fibers, crosslinked with PLLA, could enhance the compressive strength of the scaffold about four times. Human marrow mesenchymal stem cells (MSCs) culture showed that the reinforced nHACP scaffolds were more cytocompatible than that without reinforcement. The crosslinks hardly affected the cytocompatibility of the reinforced scaffolds. The results suggested that the reinforced scaffolds (DCC crosslinked) might be a promising candidate for bone-tissue engineering. PMID:16245287

  18. Synthesis and biological evaluation of chitin hydrogel/nano ZnO composite bandage as antibacterial wound dressing.

    PubMed

    Kumar, P T Sudheesh; Lakshmanan, Vinoth-Kumar; Biswas, Raja; Nair, Shantikumar V; Jayakumar, R

    2012-12-01

    We developed chitin hydrogel/nano ZnO composite bandages using chitin hydrogel and ZnO nanoparticles (nZnO). The homogenized mixture of chitin hydrogel and nZnO was freeze-dried to obtain micro-porous composite bandages. The prepared nanocomposite bandages were characterized using FT-IR, XRD and SEM. In addition, blood clotting, antibacterial, swelling, cytocompatibility and cell attachment capability of the prepared nanocomposite bandages were evaluated. The nanocomposite bandages showed enhanced swelling, blood clotting and antibacterial activity. The incorporation of nZnO helped to attain antibacterial activity. Cytocompatibility studies were carried out using human dermal fibroblast (HDF) cells proved the non-toxic nature of the composite bandages. HDF cell attachment and infiltration analysis showed that the cells were attached and penetrated into the interior (250 microm) of the nanocomposite bandages. These studies revealed that, this nanocomposite can be used for burn, diabetic and chronic wound defects. PMID:23029997

  19. Stable hydrogen-isotope ratios in beetle chitin: preliminary European data and re-interpretation of North American data

    NASA Astrophysics Data System (ADS)

    Gröcke, Darren R.; Schimmelmann, Arndt; Elias, Scott; Miller, Randall F.

    2006-08-01

    Beetle exoskeletons contain chitin, a poly amino-sugar that is biosynthesized incorporating hydrogen isotopes from diet and water. As the stable isotope ratios D/H (or 2H/ 1H, expressed as ? D values) of precipitation and diet are jointly influenced by climate, the biochemically recorded hydrogen-isotope ratio in fossil beetle exoskeleton has the potential to be used for paleoclimatic reconstruction. New ? D data from modern beetles are presented as a preliminary database for Europe, with a re-evaluation of earlier North American data. We present correlated matrices of ? D values in modern beetle chitin and modern precipitation to demonstrate the concept. We review the pertinent literature to highlight the history, utility, and likely future research directions for the use of chitin's stable isotopes in entomological paleoclimatology.

  20. Effect of Chitin Isolated from Crustaceans and Cephalopods on Denaturation of Fish Myofibrillar Protein and the State of Water during Frozen Storage

    NASA Astrophysics Data System (ADS)

    Arredondo Romero, Eduardo; Nakamura, Yukio; Yamashita, Yasumitsu; Ichikawa, Hisashi; Goto, Shingi; Osatomi, Kiyoshi; Nozaki, Yukinori

    From the point of view of utilization of shells as a waste product of fishery industry, the cryoprotective effect of chitin made from shell of crustaceans (Japanese fan lobster and Japanese swimming crab) and cartilage of cephalopods (spear squid) are studied. Chitin from the shells and cartilage were added to lizard fish myofibrils, and the changes of unfrozen water in myofibrils and ATPase activity of myofibrillar protein were observed during frozen storage at -250°C for 120days. The amount of unfrozen water were increased by addition of three kinds of chitin, and decreased moderately during forzen storage. Whereas, in the chitin free sample, the amount of unfrozen water were decreased rapidly during frozen storage. Changes of ATPase activity of samples showed similar tendency to that of the amount of unfrozen water. The present moderate cryoprotective effect of chitin and data of unfrozen water and ATPase activity of myofibrillar protein suggest the importance of the amount of unfrozen water in frozen matrix.

  1. In vitro lipid digestion of chitin nanocrystal stabilized o/w emulsions.

    PubMed

    Tzoumaki, Maria V; Moschakis, Thomas; Scholten, Elke; Biliaderis, Costas G

    2013-01-01

    Chitin nanocrystals (ChN) have been shown to form stable Pickering emulsions. These oil-in-water emulsions were compared with conventional milk (whey protein isolate, WPI, and sodium caseinate, SCn) protein-stabilized emulsions in terms of their lipid digestion kinetics using an in vitro enzymatic protocol. The kinetics of fatty acid release were evaluated as well as the change in oil droplet size of the respective emulsions during lipid digestion. The interfacial pressure was measured by addition of the duodenal components using drop tensiometry and the electrical charge of the oil droplets was also assessed, in an attempt to relate the interfacial properties with the stability of the emulsions towards lipolysis. Lipid hydrolysis in the ChN-stabilized emulsion was appreciably slower and the plateau values of the total concentration of fatty acids released were much lower, compared to the WPI- and SCn-stabilized emulsions. Moreover, the ChN-stabilized emulsions were relatively stable to coalescence during lipid digestion, whereas the WPI- and SCn-stabilized emulsions exhibited a significant increase in their droplet size. On the other hand, no major differences were shown among the different emulsion samples in terms of their interfacial properties. The increased stability of the ChN-stabilized emulsions towards lipolysis could be attributed to several underlying mechanisms: (i) strong and irreversible adsorption of the chitin nanocrystals at the interface that might inhibit an extensive displacement of the solid particles by bile salts and lipase, (ii) network formation by the nanocrystals in the bulk (continuous) phase that may reduce lipid digestion kinetics, and (iii) the ability of chitin, and consequently of ChNs, to impair pancreatic lipase activity. The finding that ChNs can be used to impede lipid digestion may have important implications for the design and fabrication of structured emulsions with controlled lipid digestibility that could provide the basis for the development of novel products that may promote satiety, reduce caloric intake and combat obesity. PMID:23064096

  2. A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties.

    PubMed

    Tynyakov, Jenny; Bentov, Shmuel; Abehsera, Shai; Yehezkel, Galit; Roth, Ziv; Khalaila, Isam; Weil, Simy; Berman, Amir; Plaschkes, Inbar; Tom, Moshe; Aflalo, Eliahu D; Sagi, Amir

    2015-11-01

    Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers-Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was also found to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation. PMID:26385331

  3. Chitin extraction from crab shells by Bacillus bacteria. Biological activities of fermented crab supernatants.

    PubMed

    Hajji, Sawssen; Ghorbel-Bellaaj, Olfa; Younes, Islem; Jellouli, Kemel; Nasri, Moncef

    2015-08-01

    Crab shells waste were fermented using six protease-producing Bacillus species (Bacillus subtilis A26, Bacillus mojavensis A21, Bacillus pumilus A1, Bacillus amyloliquefaciens An6, Bacillus licheniformis NH1 and Bacillus cereus BG1) for the production of chitin and fermented-crab supernatants (FCSs). In medium containing only crab shells, the highest demineralization DM was obtained with B. licheniformis NH1 (83±0.5%) and B. pumilus A1 (80±0.6%), while the highest deproteinization (DP) was achieved with A1 (94±1%) followed by NH1 (90±1.5%) strains. Cultures conducted in medium containing crab shells waste supplemented with 5% (w/v) glucose, were found to remarkably promote demineralization efficiency, and enhance slightly deproteinization rates. FTIR spectra of chitins showed the characteristics bands of ?-chitin. FCSs showed varying degrees of antioxidant activities which were in a dose-dependent manner (p<0.01). In fact, FCS produced by B. amyloliquefaciens An6 exhibited the highest DPPH free radical-scavenging activity (92% at 4 mg/ml), while the lowest hydroxyl radical-scavenging activity (60% at 4 mg/ml) was obtained with B. subtilis A26 hydrolysates. However, the highest reducing power (OD700nm=2 at 0.5 mg/ml) was obtained by B.amyloliquefaciens An6 hydrolysates. These results suggest that crab hydrolysates are good sources of natural antioxidants. Further, FCSs were found to exhibit antibacterial activity against Gram-positive and Gram-negative bacteria. PMID:25910648

  4. A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

    PubMed Central

    Zhou, Hui; Zhan, Jixun; Watanabe, Kenji; Xie, Xinkai; Tang, Yi

    2008-01-01

    Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Using a small molecule mimic of the natural hexaketide starter unit, we reconstituted the entire repertoire of PKS13 activities in vitro, including starter-unit selection, iterative condensation, regioselective C2–C7 cyclization, and macrolactone formation. PKS13 synthesized both natural 14-membered and previously uncharacterized 16-membered resorcylic acid lactones, indicating relaxed control in both iterative elongation and macrocyclization. PKS13 exhibited broad starter-unit specificities toward fatty acyl-CoAs ranging in sizes between C6 and C16 and displayed the highest activity toward decanoyl-CoA. PKS13 was shown to be active in Escherichia coli and synthesized numerous alkyl pyrones and alkyl resorcylic esters without exogenously supplied precursors. We demonstrated that PKS13 can interact with E. coli fatty acid biosynthetic machinery and can be primed with fatty-acyl ACPp at low-micromolar concentrations. PKS13 synthesized new polyketides in E. coli when the culture was supplemented with synthetic precursors, showcasing its utility in precursor-directed biosynthesis. PKS13 is therefore a highly versatile polyketide macrolactone synthase that is useful in the engineered biosynthesis of polyketides, including resorcylic acid lactones that are not found in nature. PMID:18427109

  5. Chelating ability of chitinous materials from Streptomyces, Mucor rouxii, Phycomyces blakesleeanus, and Choanephora cucurbitarum

    SciTech Connect

    Muzzarelli, R.A.A.; Tanfani, F.; Emanuelli, M.

    1981-08-01

    The mycelia of Streptomyces, Mucor rouxii, Phycomyces blakesleeanus, and Choanephora cucurbitarum, after treatment either with 40% NaOH at 100/sup 0/C for 3-4 h or 0.01 M NaOH for 60 min, were studied by IR spectrometry, to assess their chitin and chitosan contents, and used to collect Cr, Mn, Co, Ni, Cu, Zn, Hg, and Pb from 0.5 mM solutions at 20 and 60/sup 0/C. The results reflect the chelating ability of chitosan and are especially high for the Streptomyces inactivated material, which is available in large amounts as a waste of industrial fermentations.

  6. HYDROGEN PEROXIDE INDUCED OXIDATION OF PEROXISOMAL MALATE SYNTHASE AND CATALASE.

    E-print Network

    Simha, Rahul

    Page 1 HYDROGEN PEROXIDE INDUCED OXIDATION OF PEROXISOMAL MALATE SYNTHASE AND CATALASE. Pria Anand1 locations within the proteins' structures. Key words: biotin hydrazide, carbonylation, catalase, hydrogen peroxide, glyoxysome, MALDI- TOF MS, malate synthase, mass spectroscopy, peroxisome, protein oxidation

  7. Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

    PubMed

    Sato, Tsutomu; Yamaga, Hiroaki; Kashima, Shoji; Murata, Yusuke; Shinada, Tetsuro; Nakano, Chiaki; Hoshino, Tsutomu

    2013-05-10

    Basic enzyme: The tetraprenyl-?-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-?-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. PMID:23554321

  8. Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase

    E-print Network

    Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase subunits/UPMC, Institut de Biologie Physico-Chimique, Paris, France The (C)F1 sector from Hþ -ATP synthases comprises five; Published online 26 July 2007 Subject Categories: proteins Keywords: ATP synthase assembly; Chlamydomonas

  9. Chitin from Penaeus merguiensis via microbial fermentation processing and antioxidant activity.

    PubMed

    Sedaghat, Fatemeh; Yousefzadi, Morteza; Toiserkani, Hojjat; Najafipour, Sohrab

    2016-01-01

    The ability of three proteases producing bacteria such as Pseudomonas aeruginosa, Serratia marcescens, and Bacillus pumilus on the demineralization and deproteinzation efficiency of shrimp waste, for chitin extraction, was investigated. Statistical analysis of data was showed a significant difference between the percentage of demineralization and deproteinization in different bacteria species (p<0.05). The highest deproteinization (74.76%) and demineralization rate (78.46%) was obtained with P. aeruginosa and the lowest was observed in the treatment of S. marcescens. Then, chitin was converted to chitosan by deacetylation in the presence of NaOH 50%. The antioxidant activity of chitosan solution was determined using different tests. The highest activity (DO 700nm=0.74, DO 695nm=0.31) was observed for chitosan sample at concentration of 1000?g/ml. The antioxidant potential of the hydrolysates was also evaluated. The highest reducing power in a volume of 400?l hydrolysate of S. marcescens and the highest total antioxidant capacity in a volume 100?l hydrolysate of B. pumilus were observed. These results indicated that the P. aeruginosa bacterium in comparison with other bacterial strains, higher ability to remove proteins and mineral from shrimp waste. Therefore, the use of this bacterium is recommended for protein and mineral removal from marine crustaceans. PMID:26519877

  10. The fungicidal properties of the carbon materials obtained from chitin and chitosan promoted by copper salts.

    PubMed

    Ilnicka, Anna; Walczyk, Mariusz; Lukaszewicz, Jerzy P

    2015-07-01

    Renewable raw materials chitin and chitosan (N-deacetylated derivative of chitin) were subjected to action of different copper modifiers that were carbonized in the atmosphere of the N2 inert gas. As a result of the novel manufacturing procedure, a series of carbon materials was obtained with developed surface area and containing copper derivatives of differentiated form, size, and dispersion. The copper modifier and manufacturing procedure (concentration, carbonization temperature) influence the physical-chemical and fungicide properties of the carbons. The received carbons were chemically characterized using several methods like low-temperature adsorption of nitrogen, X-ray diffraction analysis, scanning electron microscopy, cyclic voltammetry, elemental analysis, and bioassay. Besides chemical testing, some biological tests were performed and let to select carbons with the highest fungicidal activity. Such carbons were characteristic of the specific form of copper derivatives occurring in them, i.e., nanocrystallites of Cu(0) and/or Cu2O of high dispersion on the surface of carbon. The carbons may find an application as effective contact fungistatic agents in cosmetology, medicine, food industry, etc. PMID:25953537

  11. Electrospun chitosan-based nanocomposite mats reinforced with chitin nanocrystals for wound dressing.

    PubMed

    Naseri, Narges; Algan, Constance; Jacobs, Valencia; John, Maya; Oksman, Kristiina; Mathew, Aji P

    2014-08-30

    The aim of this study was to develop electrospun chitosan/polyethylene oxide-based randomly oriented fiber mats reinforced with chitin nanocrystals (ChNC) for wound dressing. Microscopy studies showed porous mats of smooth and beadless fibers with diameters between 223 and 966 nm. The addition of chitin nanocrystals as well as crosslinking had a positive impact on the mechanical properties of the mats, and the crosslinked nanocomposite mats with a tensile strength of 64.9 MPa and modulus of 10.2 GPa were considered the best candidate for wound dressing application. The high surface area of the mats (35 m(2)g(-1)) was also considered beneficial for wound healing. The water vapor transmission rate of the prepared mats was between 1290 and 1,548 gm(-2)day(-1), and was in the range for injured skin or wounds. The electrospun fiber mats showed compatibility toward adipose derived stem cells, further confirming their potential use as wound dressing materials. PMID:24815394

  12. Clinical and environmental genotypes of Vibrio vulnificus display distinct, quorum-sensing-mediated, chitin detachment dynamics.

    PubMed

    Phippen, Britney L; Oliver, James D

    2015-11-01

    The ability for bacteria to attach to and detach from various substrata is important for colonization, survival and transitioning to new environments. An opportunistic human pathogen, Vibrio vulnificus, can cause potentially fatal septicemia after ingestion of undercooked seafood. Based on genetic polymorphisms, strains of this species are subtyped into clinical (C) and environmental (E) genotypes. Vibrio vulnificus readily associates with chitin, thus we investigated chitin detachment dynamics in these disparate genotypes. We found that C-genotypes detach significantly more than E-genotypes after 24 hours in aerobic as well as anaerobic conditions. Furthermore, expression of genes involved in type IV pilin production was significantly downregulated in C-genotypes compared to E-genotypes, suggesting an importance in detachment. Interestingly, gbpA, a gene that has been shown to be important in host colonization in V. cholerae, was upregulated in the C-genotypes during detachment. Additionally, we found that C-genotypes detached to a greater extent, and produced more quorum-sensing (QS) autoinducer-2 molecules relative to E-genotypes, which suggests a role for QS in detachment. These findings suggest that for V. vulnificus, QS-mediated detachment may be a potential mechanism for transitioning into a human host for C-genotypes, while facilitating E-genotype maintenance in the estuarine environment. PMID:26377182

  13. Chitin-incorporated poly(ethylene oxide)-based nanocomposite electrolytes for lithium batteries.

    PubMed

    Stephan, A Manuel; Kumar, T Prem; Kulandainathan, M Anbu; Lakshmi, N Angu

    2009-02-19

    Nanocomposite polymer electrolytes (NCPE), with different proportions of poly(ethylene oxide)/LiClO(4)/chitin were prepared by a hot press method. Nanochitin, a biopolymer, poly(beta-(1-->4)-N acetyl-d-glucosamine) was incorporated as a filler in poly(ethylene oxide) (PEO). The ionic conductivity of the composite polymer electrolytes was enhanced by one order upon addition of nanochitin. The lithium transference number, t(Li)(+), was increased from 0.24 to 0.51 upon chitin addition. The membranes were subjected to scanning electron microscopy, thermogravimetric-differential thermal analysis, differential scanning calorimetry, ionic conductivity, and Fourier transform infrared (FTIR) spectroscopy analysis. The free volume V(f) was probed by positron annihilation lifetime spectroscopy studies at 30 degrees C. Li/NCPE/Li symmetric cells were assembled, and the thickness of the solid electrolyte interface as a function of time was analyzed. This paper also describes FTIR spectroscopic studies of the interface between lithium metal and NCPE, which suggests that the surface chemistry of lithium electrodes in contact with NCPE is dominated by compounds with C-N-Li and C-O-Li bonding. PMID:19161288

  14. Haem synthase and cobalt porphyrin synthase in various micro-organisms

    PubMed Central

    Porra, R. J.; Ross, B. D.

    1965-01-01

    1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co2+ ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co2+ ions into these two porphyrins is rapid. These extracts are unable to insert Mn2+, Zn2+, Mg2+ or Cu2+ ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co2+ and Fe2+ ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co2+ ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents. PMID:14340046

  15. Producing dicarboxylic acids using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  16. Producing dicarboxylic acids using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  17. Lessons from 455 Fusarium polyketide synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

  18. Effect of Chitin Isolated from Crustaceans and Cephalopods on Denaturation of Fish Myofibrillar Protein and the State of Water during Frozen Storage

    NASA Astrophysics Data System (ADS)

    Yamashita, Yasumitsu; Zhang, Nong; Nozaki, Yukinori

    The effect of chitin hydrolysate made from shell of crustaceans and cartilage of cephalopods on the denaturation and the state of water of lizard fish myofibrillar protein (Mf) during frozen storage at -250°C for 120 days were investigated. 5.0% chitin hydrolysate (dried matter) added Mf samples were frozen at -25°C and storage for 120 days, and the change of inactivation of Mf Ca-ATPase and the change of unfrozen water in Mf were examined during the frozen storage. The decrease of Mf Ca-ATPase during frozen storage were slow by addition of chitin hydrolysate. The amount of unfrozen water in Mf were increased by addition of chitin hydrolysate, and decreaced gradually during frozen storage. On the other hand, the amount of unfrozen water of the control were decreaced suddenly during frozen storage. Above results suggest that chitin hydrolysate has cryoprotective effect on Mf. It is guessed that the mechanism of cryoprotective effect of chitin hydrolysate may be caused by an interaction between hydration sphere of Mf and equatorial OH groups in the molecular structure of chitin hydrolysate.

  19. Mitochondrial ?-Cyanoalanine Synthase Is Essential for Root Hair Formation in Arabidopsis thaliana[W

    PubMed Central

    García, Irene; Castellano, José María; Vioque, Blanca; Solano, Roberto; Gotor, Cecilia; Romero, Luis C.

    2010-01-01

    Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by ?-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial ?-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial ?-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development. PMID:20935247

  20. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

    PubMed Central

    Atkinson, Sarah C.; Dogovski, Con; Dobson, Renwick C. J.; Perugini, Matthew A.

    2012-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP_354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40?Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents. PMID:22949190

  1. A shared mechanism of defense against predators and parasites: chitin regulation and its implications for life-history theory

    PubMed Central

    Beckerman, Andrew P; de Roij, Job; Dennis, Stuart R; Little, Tom J

    2013-01-01

    Defenses against predators and parasites offer excellent illustrations of adaptive phenotypic plasticity. Despite vast knowledge about such induced defenses, they have been studied largely in isolation, which is surprising, given that predation and parasitism are ubiquitous and act simultaneously in the wild. This raises the possibility that victims must trade-off responses to predation versus parasitism. Here, we propose that arthropod responses to predators and parasites will commonly be based on the endocrine regulation of chitin synthesis and degradation. The proposal is compelling because many inducible defenses are centered on temporal or spatial modifications of chitin-rich structures. Moreover, we show how the chitin synthesis pathway ends in a split to carapace or gut chitin, and how this form of molecular regulation can be incorporated into theory on life-history trade-offs, specifically the Y-model. Our hypothesis thus spans several biological scales to address advice from Stearns that “Endocrine mechanisms may prove to be only the tip of an iceberg of physiological mechanisms that modulate the expression of genetic covariance”. PMID:24455141

  2. Distribution of Calcium and Chitin in the Tardigrade Feeding Apparatus in Relation to its Function and Morphology.

    PubMed

    Guidetti, Roberto; Bonifacio, Alois; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

    2015-08-01

    The cuticular portion of the tardigrade feeding apparatus is a complex structure that can be schematically divided into four parts: a buccal ring, a buccal tube, a stylet system (formed by two piercing stylets, each within a stylet coat, and two stylet supports), and the lining of a myoepithelial sucking pharynx. To better understand the function and evolution of the feeding apparatus, the morpho-functional traits and chemical composition of the structures forming the feeding apparatuses of eight different species of tardigrades were analyzed. These eight species are representative of almost all main phylogenetic lineages of the phylum. The calcium and chitin in the feeding apparatus were examined by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, energy dispersive X-ray spectroscopy, and Raman microspectroscopy (Raman). In all species, the feeding apparatus had been subjected to biomineralization due to CaCO3 encrustations organized in the crystalline form of aragonite. Aragonite and chitin are present in different concentrations in the feeding apparatus according to the structures and species considered. Generally, where the structures are rigid there is more aragonite than chitin, and vice versa. The buccal tube and piercing stylets are rich in calcium, with the piercing stylets apparently composed exclusively of aragonite. In eutardigrades, chitin is in higher concentration in the structures subject to higher mechanical stresses, such as the crests of the buccal crown and the condyles of the stylet furca. PMID:25857526

  3. Optimization of medium components for production of chitin deacetylase by Bacillus amyloliquefaciens Z7, using response surface methodology

    PubMed Central

    He, Yuanhao; Xu, Jianping; Wang, Shengjie; Zhou, Guoying; Liu, Junang

    2014-01-01

    Plackett–Burman design and Box–Behnken response surface methodology (RSM) was employed to optimize the medium components for the chitin deacetylase (CDA) activity from Bacillus amyloliquefaciens Z7. Plackett–Burman design was applied to determine the specific medium components affecting CDA activity and found that starch, chitin and MgSO4 were critical in augmenting CDA activity. These significant parameters were further optimized using Box–Behnken RSM and the optimum concentrations of starch, chitin and MgSO4 were found to be 24.4, 8.8 and 0.19 g/L, respectively. The optimum medium composition was chitin 8.8 g/L, starch 24.4 g/L, yeast extract 10g/L, MgSO4 0.19 g/L, K2HPO4 0.3 g/L and NaCl 5 g/L. Under these optimal conditions, the CDA activity of Bacillus amyloliquefaciens Z7 increased distinctly from 18.75 to 27.48 U/mL (46.6% increase in total yield).

  4. Injectable Chitin-Poly(?-caprolactone)/Nanohydroxyapatite Composite Microgels Prepared by Simple Regeneration Technique for Bone Tissue Engineering.

    PubMed

    Arun Kumar, R; Sivashanmugam, A; Deepthi, S; Iseki, Sachiko; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

    2015-05-13

    Injectable gel systems, for the purpose of bone defect reconstruction, have many advantages, such as controlled flowability, adaptability to the defect site, and increased handling properties when compared to the conventionally used autologous graft, scaffolds, hydroxyapatite blocks, etc. In this work, nanohydroxyapatite (nHAp) incorporated chitin-poly(?-caprolactone) (PCL) based injectable composite microgels has been developed by a simple regeneration technique for bone defect repair. The prepared microgel systems were characterized using scanning electron microscope (SEM), Fourier transformed infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The composite microgel, with the incorporation of nHAp, showed an increased elastic modulus and thermal stability and had shear-thinning behavior proving the injectability of the system. The protein adsorption, cytocompatibility, and migration of rabbit adipose derived mesenchymal stem cells (rASCs) were also studied. Chitin-PCL-nHAp microgel elicited an early osteogenic differentiation compared to control gel. The immunofluorescence studies confirmed the elevated expression of osteogenic-specific markers such as alkaline phosphatase, osteopontin, and osteocalcin in chitin-PCL-nHAp microgels. Thus, chitin-PCL-nHAp microgel could be a promising injectable system for regeneration of bone defects which are, even in deeper planes, irregularly shaped and complex in nature. PMID:25893690

  5. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B. (Pullman, WA); Burke, Charles C. (Moscow, ID); Wildung, Mark R. (Colfax, WA)

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, 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 large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  6. The affinity purification and characterization of ATP synthase complexes from mitochondria

    PubMed Central

    Runswick, Michael J.; Bason, John V.; Montgomery, Martin G.; Robinson, Graham C.; Fearnley, Ian M.; Walker, John E.

    2013-01-01

    The mitochondrial F1-ATPase inhibitor protein, IF1, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the ?-helical inhibitory region of the bound IF1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the ?-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region. PMID:23407638

  7. Biochemical and Structural Basis for Inhibition of Enterococcus faecalis Hydroxymethylglutaryl-CoA Synthase, mvaS, by Hymeglusin

    SciTech Connect

    Skaff, D. Andrew; Ramyar, Kasra X.; McWhorter, William J.; Barta, Michael L.; Geisbrecht, Brian V.; Miziorko, Henry M.

    2012-07-25

    Hymeglusin (1233A, F244, L-659-699) is established as a specific {beta}-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 {angstrom}) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

  8. Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin?induction

    PubMed Central

    Ni, Xiangyang; Westpheling, Janet

    1997-01-01

    The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5? and 3? deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at ?10, ?12, ?32, ?33, ?35, and ?37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around ?10 (TATTCT) and ?35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression. PMID:9371809

  9. Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System

    PubMed Central

    Kharade, Sampada S.

    2014-01-01

    Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS. PMID:24363341

  10. Influence of carboxymethyl chitin on stability and biocompatibility of 3D nanohydroxyapatite/gelatin/carboxymethyl chitin composite for bone tissue engineering.

    PubMed

    Sagar, Nitin; Soni, Vivek P; Bellare, Jayesh R

    2012-04-01

    A novel three-dimensional (3D) scaffold has been developed from the unique combination of nanohydroxyapatite/gelatin/carboxymethyl chitin (n-HA/gel/CMC) for bone tissue engineering by using the solvent-casting method combined with vapor-phase crosslinking and freeze-drying. The surface morphology and physiochemical properties of the scaffold were investigated by dissolvability test, infrared absorption spectra (IR), X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscope (SEM), mechanical testing, and soaking in simulated body fluid (SBF). An optimized (composition and processing parameters) ratio of n-HA:gel:CMC (1:2:1), exhibited ideal porous structure with regular interconnected pores (75-250 ?m) and higher mechanical strength. Result suggested that the divalent (Ca(++)), carboxyl (COO(-)), amino (NH4(+)), and phosphate (PO4(3-)) groups created favorable ionic interactions which facilitated structural stability and integrity of the composite scaffold. The SBF soaking experiment confirmed the apatite nucleation ability, induced by CMC incorporation. Furthermore, hemocompatibility (hemolysis, platelet adhesion, and protein adsorption) and biocompatibility with MG63 osteoblast cells (MTT assay, cell morphology, and confocal studies from within the 3D scaffold) indicated that the structural and dimensional stability of composite scaffold provided an optimal mechanosensory environment for enhancement of cell adhesion, proliferation, and network formation. The n-HA/gel/CMC composite, therefore, may serve as a promising composite scaffold for guided bone regeneration. PMID:22323281

  11. Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase.

    PubMed Central

    Maines, M D; Kappas, A

    1976-01-01

    Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism. PMID:826907

  12. Identification and development of mPGES-1 inhibitors: where we are at?

    PubMed Central

    Chang, Hui-Hua; Meuillet, Emmanuelle J

    2011-01-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal synthase responsible for the synthesis of the pro-tumorigenic prostaglandin E2 (PGE2). mPGES-1 is overexpressed in a wide variety of cancers. Since its discovery in 1997 by Bengt Samuelsson and collaborators, the enzyme has been the object of over 200 peer-reviewed articles. Although today mPGES-1 is considered a validated and promising therapeutic target for anticancer drug discovery, challenges in inhibitor design and selectivity are such that up to this date there are only a few published records of small-molecule inhibitors targeting the enzyme and exhibiting some in vivo anticancer activity. This review summarizes the structures, and the in vitro and in vivo activities of these novel mPGES-1 inhibitors. Challenges that have been encountered are also discussed. PMID:22023034

  13. Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells.

    PubMed

    Armour, K E; Ralston, S H

    1998-02-01

    The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone. PMID:9449657

  14. Ectopic ATP synthase facilitates transfer of HIV-1 from antigen-presenting cells to CD4+ target cells

    PubMed Central

    Yavlovich, Amichai; Viard, Mathias; Zhou, Ming; Veenstra, Timothy D.; Wang, Ji Ming; Gong, Wanghua; Heldman, Eliahu; Blumenthal, Robert

    2012-01-01

    Antigen-presenting cells (APCs) act as vehicles that transfer HIV to their target CD4+ cells through an intercellular junction, termed the virologic synapse. The molecules that are involved in this process remain largely unidentified. In this study, we used photoaffinity labeling and a proteomic approach to identify new proteins that facilitate HIV-1 transfer. We identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between APCs and CD4+ target cells. Monoclonal antibodies against the ?-subunit of ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4+ target cells. Likewise, the specific inhibitors of ATPase, citreoviridin and IF1, completely blocked APC-mediated transfer of HIV-1 at the APC-target cell interaction step. Confocal fluorescent microscopy showed localization of extracellular ATP synthase at junctions between APC and CD4+ target cells. We conclude that ectopic ATP synthase could be an accessible molecular target for inhibiting HIV-1 proliferation in vivo. PMID:22753871

  15. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  16. Fluconazole loaded chitin nanogels as a topical ocular drug delivery agent for corneal fungal infections.

    PubMed

    Mohammed, Nishil; Rejinold, N Sanoj; Mangalathillam, Sabitha; Biswas, Raja; Nair, Shantikumar V; Jayakumar, R

    2013-09-01

    Poor bioavailability of antifungal drugs due to the various protective mechanisms of the eye is a serious concern for the treatment of corneal fungal infections in today's world. The use of nanosystems that can improve the bioavailability of these antifungal drugs is relatively a new idea being conceived and here we have synthesized fluconazole loaded chitin nanogels (Flu-CNGs) which can be used for the treatment of corneal fungal infections. These nanogels were characterized using DLS, Zeta potential, SEM, FTIR and TG/DTA. The prepared Flu-CNGs have controlled release pattern which is ideal for the continuous availability of fluconazole over a longer period of time for an effective fungal treatment. Flu-CNGs are haemocompatible, cytocompatible and also showed very good cell uptake in human dermal fibroblast cells and penetration to the deeper sections of the porcine cornea with no signs of destruction or inflammation to corneal cells as shown in ex vivo permeation studies. PMID:23980500

  17. Depolymerization and de-N-acetylation of chitin oligomers in hydrochloric acid.

    PubMed

    Einbu, Aslak; Vårum, Kjell M

    2007-01-01

    The monosaccharide 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) has recently drawn much attention in relation to its use to treat or prevent osteoarthritis in humans. Glucosamine is prepared from chitin, a process that is performed in concentrated acid, such as hydrochloric acid. This process involves two acid-catalyzed processes, that is, the hydrolysis of the glycosidic linkages (depolymerization) and of the N-acetyl linkages (de-N-acetylation). The depolymerization reaction has previously been found to be much faster compared to the deacetylation, with the consequence that the chitin chain will first be hydrolyzed to the monomer 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) which is subsequently deacetylated. We have found that the chitin disaccharide GlcNAc(1-->4)GlcNAc could be completely hydrolyzed to the monosaccharide GlcNAc with negligible concomitant de-N-acetylation, and the chitin disaccharide and monosaccharide were further used to study the depolymerization reaction and the de-N-acetylation reaction, respectively. The reactions were performed in hydrochloric acid as a function of acid concentration (3-12 M) and temperature (20-35 degrees C), and 1H-NMR spectroscopy was used to monitor the reaction rates. The 1H NMR spectrum of GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The glucofuranosyl oxazolinium (3) ion was found to exist in equilibrium with the alpha- and beta-anomers of the pyranose form of GlcNAc, where 3 was present in half the total molar concentrations of the two anomeric forms of GlcNAc. At lower acid concentration (3-6 M), only trace concentrations of 3 could be detected. The rate of de-N-acetylation of GlcNAc was determined as a function of hydrochloric acid concentration, showing a maximum at 6 M and decreasing by a factor of 2 upon decreasing or increasing the acid concentration to 3 or 12 M. The activation energy for hydrolysis of the N-acetyl linkage of GlcNAc was determined to be 102 +/- 7, 116 +/- 8, and 110 +/- 8 kJ mol(-1) at 3, 6, and 12 M hydrochloric acid concentration, respectively. The results are in accordance with the proposed SN2 reaction mechanism of the acid-catalyzed hydrolysis of the N-acetyl linkage where the rate-limiting step is the addition of water to the carbonium ion. The 1H NMR spectrum of the dimer GlcNAc-GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The rate of the acid-catalyzed cleavage of the glycosidic linkage of the dimer was determined as a function of hydrochloric acid concentration, showing a 6-fold increase from 3 to 6 M HCl concentration and a further 6-fold increase from 6 to 12 M HCl concentration, in contrast to the much smaller effect of acid concentration on the deacetylation reaction. Activation energy for hydrolysis of the glycosidic linkage of GlcNAc-GlcNAc was determined to be 110 +/- 6, 111 +/- 6, and 112 +/- 4 kJ mol(-1) at 3, 6 and 12 M hydrochloric acid concentration, respectively, that is, very similar to the activation energies determined for the deacetylation reaction. The results are in accordance with the proposed SN1 reaction mechanism of the acid-catalyzed hydrolysis of the glycosidic linkage, where the rate-limiting step is the formation of the carbonium ion. PMID:17206822

  18. Prominent reinforcing effect of chitin nanocrystals on electrospun polydioxanone nanocomposite fiber mats.

    PubMed

    Zhu, Lei; Liang, Kai; Ji, Yali

    2015-04-01

    The ultra-strong nanocomposite fiber mats based on biodegradable polydioxanone (PDO) and chitin nanocrystals (ChiNCs) were successfully prepared by means of electrospinning. The ChiNCs are uniformly dispersed in the PDO matrix and mostly oriented along fiber long axis, resulting in a significant improvement in mechanical property. Moreover, the introduction of ChiNCs led to the increase of the glass-transition temperature (Tg) and thermal decomposition temperature (Td) of PDO elucidated by thermal analyses. In addition, the loading of ChiNCs caused very different In vitro degradation behavior compared to neat PDO fiber mat. Furthermore, in vitro cell culture results indicated that the addition of ChiNCs improved the cellular adhesion and proliferation. PMID:25598072

  19. Effects of Oral Administration of Chitin Nanofiber on Plasma Metabolites and Gut Microorganisms.

    PubMed

    Azuma, Kazuo; Izumi, Ryotaro; Kawata, Mari; Nagae, Tomone; Osaki, Tomohiro; Murahata, Yusuke; Tsuka, Takeshi; Imagawa, Tomohiro; Ito, Norihiko; Okamoto, Yoshiharu; Morimoto, Minoru; Izawa, Hironori; Saimoto, Hiroyuki; Ifuku, Shinsuke

    2015-01-01

    The aim of this study was to examine the effects of oral administration of chitin nanofibers (CNFs) and surface-deacetylated (SDA) CNFs on plasma metabolites using metabolome analysis. Furthermore, we determined the changes in gut microbiota and fecal organic acid concentrations following oral administrations of CNFs and SDACNFs. Healthy female mice (six-week-old) were fed a normal diet and administered tap water with 0.1% (v/v) CNFs or SDACNFs for 28 days. Oral administration of CNFs increased plasma levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and serotonin (5-hydroxytryptamine, 5-HT). Oral administration of SDACNFs affected the metabolisms of acyl-carnitines and fatty acids. The fecal organic level analysis indicated that oral administration of CNFs stimulated and activated the functions of microbiota. These results indicate that oral administration of CNFs increases plasma levels of ATP and 5-HT via activation of gut microbiota. PMID:26378523

  20. Effects of Oral Administration of Chitin Nanofiber on Plasma Metabolites and Gut Microorganisms

    PubMed Central

    Azuma, Kazuo; Izumi, Ryotaro; Kawata, Mari; Nagae, Tomone; Osaki, Tomohiro; Murahata, Yusuke; Tsuka, Takeshi; Imagawa, Tomohiro; Ito, Norihiko; Okamoto, Yoshiharu; Morimoto, Minoru; Izawa, Hironori; Saimoto, Hiroyuki; Ifuku, Shinsuke

    2015-01-01

    The aim of this study was to examine the effects of oral administration of chitin nanofibers (CNFs) and surface-deacetylated (SDA) CNFs on plasma metabolites using metabolome analysis. Furthermore, we determined the changes in gut microbiota and fecal organic acid concentrations following oral administrations of CNFs and SDACNFs. Healthy female mice (six-week-old) were fed a normal diet and administered tap water with 0.1% (v/v) CNFs or SDACNFs for 28 days. Oral administration of CNFs increased plasma levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and serotonin (5-hydroxytryptamine, 5-HT). Oral administration of SDACNFs affected the metabolisms of acyl-carnitines and fatty acids. The fecal organic level analysis indicated that oral administration of CNFs stimulated and activated the functions of microbiota. These results indicate that oral administration of CNFs increases plasma levels of ATP and 5-HT via activation of gut microbiota. PMID:26378523

  1. Self-assembled cardanol azo derivatives as antifungal agent with chitin-binding ability.

    PubMed

    Mahata, Denial; Mandal, Santi M; Bharti, Rashmi; Gupta, Vinay Krishna; Mandal, Mahitosh; Nag, Ahindra; Nando, Golok B

    2014-08-01

    Cardanol is a non-isoprenoic phenolic lipid-mixture of distilled cashew nut shell liquid obtained from Anacardium occidentale. Herein, cardanol is purified from cashew nut shell liquid (CNSL) and synthesized to new compounds with different azo amphiphiles. These synthesized compounds are allowed to self-assembled in hydrophobic environment and checked antifungal activity against Candida albicans. Self-assembled structure of CABA showed higher antifungal activity (16?g/mL) and chitin-binding ability in comparison to CAP and CANB. Furthermore, the self-assembled azo amphiphiles are immobilized with silver ions to prepare hydrogel which showed eight folds enhanced antifungal activity. Toxicity is reduced by several folds of self-assembled or hydrogel structure in comparison to pure compounds. Thus, the self-assembled structure of amphiphiles and their hydrogels have been found to be new macromolecules of interest with potential use as antifungal drugs. PMID:24836571

  2. Biological Chitin-MOF Composites with Hierarchical Pore Systems for Air-Filtration Applications.

    PubMed

    Wisser, Dorothea; Wisser, Florian M; Raschke, Silvia; Klein, Nicole; Leistner, Matthias; Grothe, Julia; Brunner, Eike; Kaskel, Stefan

    2015-10-19

    Metal-organic frameworks (MOFs) are promising materials for gas-separation and air-filtration applications. However, for these applications, MOF crystallites need to be incorporated in robust and manageable support materials. We used chitin-based networks from a marine sponge as a non-toxic, biodegradable, and low-weight support material for MOF deposition. The structural properties of the material favor predominant nucleation of the MOF crystallites at the inside of the hollow fibers. This composite has a hierarchical pore system with surface areas up to 800?m(2) ?g(-1) and pore volumes of 3.6?cm(3) ?g(-1) , allowing good transport kinetics and a very high loading of the active material. Ammonia break-through experiments highlight the accessibility of the MOF crystallites and the adsorption potential of the composite indicating their high potential for filtration applications for toxic industrial gases. PMID:26314273

  3. Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

    PubMed

    Mravec, Jozef; Kra?un, Stjepan K; Rydahl, Maja G; Westereng, Bjørge; Miart, Fabien; Clausen, Mads H; Fangel, Jonatan U; Daugaard, Mathilde; Van Cutsem, Pierre; De Fine Licht, Henrik H; Höfte, Herman; Malinovsky, Frederikke G; Domozych, David S; Willats, William G T

    2014-12-01

    Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons. PMID:25395456

  4. Ancient pests: the season of the Santorini Minoan volcanic eruption and a date from insect chitin

    NASA Astrophysics Data System (ADS)

    Panagiotakopulu, Eva; Higham, Thomas; Sarpaki, Anaya; Buckland, Paul; Doumas, Christos

    2013-07-01

    Attributing a season and a date to the volcanic eruption of Santorini in the Aegean has become possible by using preserved remains of the bean weevil, Bruchus rufipes, pests of pulses, from the storage jars of the West House, in the Bronze Age settlement at Akrotiri. We have applied an improved pre-treatment methodology for dating the charred insects, and this provides a date of 1744-1538 BC. This date is within the range of others obtained from pulses from the same context and confirms the utility of chitin as a dating material. Based on the nature of the insect material and the life cycle of the species involved, we argue for a summer eruption, which took place after harvest, shortly after this material was transported into the West House storeroom.

  5. Ancient pests: the season of the Santorini Minoan volcanic eruption and a date from insect chitin.

    PubMed

    Panagiotakopulu, Eva; Higham, Thomas; Sarpaki, Anaya; Buckland, Paul; Doumas, Christos

    2013-07-01

    Attributing a season and a date to the volcanic eruption of Santorini in the Aegean has become possible by using preserved remains of the bean weevil, Bruchus rufipes, pests of pulses, from the storage jars of the West House, in the Bronze Age settlement at Akrotiri. We have applied an improved pre-treatment methodology for dating the charred insects, and this provides a date of 1744-1538 BC. This date is within the range of others obtained from pulses from the same context and confirms the utility of chitin as a dating material. Based on the nature of the insect material and the life cycle of the species involved, we argue for a summer eruption, which took place after harvest, shortly after this material was transported into the West House storeroom. PMID:23793358

  6. Insectivorous Bats Digest Chitin in the Stomach Using Acidic Mammalian Chitinase

    PubMed Central

    Strobel, Sara; Roswag, Anna; Becker, Nina I.; Trenczek, Tina E.; Encarnação, Jorge A.

    2013-01-01

    The gastrointestinal tract of animals is adapted to their primary source of food to optimize resource use and energy intake. Temperate bat species mainly feed on arthropods. These contain the energy-rich carbohydrate chitin, which is indigestible for the endogenous enzymes of a typical mammalian gastrointestinal tract. However, the gastrointestinal tract of bat species should be adapted to their diet and be able to digest chitin. We hypothesized that (i) European vespertilionid bat species have the digestive enzyme chitinase and that (ii) the chitinolytic activity is located in the intestine, as has been found for North American bat species. The gastrointestinal tracts of seven bat species (Pipistrellus pipistrellus, Plecotus auritus, Myotis bechsteinii, Myotis nattereri, Myotis daubentonii, Myotis myotis, and Nyctalus leisleri) were tested for chitinolytic activity by diffusion assay. Gastrointestinal tracts of P. pipistrellus, P. auritus, M. nattereri, M. myotis, and N. leisleri were examined for acidic mammalian chitinase by western blot analysis. Tissue sections of the gastrointestinal tract of P. pipistrellus were immunohistochemically analyzed to locate the acidic mammalian chitinase. Chitinolytic activity was detected in the stomachs of all bat species. Western blot analysis confirmed the acidic mammalian chitinase in stomach samples. Immunohistochemistry of the P. pipistrellus gastrointestinal tract indicated that acidic mammalian chitinase is located in the stomach chief cells at the base of the gastric glands. In conclusion, European vespertilionid bat species have acidic mammalian chitinase that is produced in the gastric glands of the stomach. Therefore, the gastrointestinal tracts of insectivorous bat species evolved an enzymatic adaptation to their diet. PMID:24019876

  7. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1?-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 ?m. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ?100 ?m, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 ?g h?1 g?1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  8. Benzophenone synthase from Garcinia mangostana L. pericarps.

    PubMed

    Nualkaew, Natsajee; Morita, Hiroyuki; Shimokawa, Yoshihiko; Kinjo, Keishi; Kushiro, Tetsuo; De-Eknamkul, Wanchai; Ebizuka, Yutaka; Abe, Ikuro

    2012-05-01

    The cDNA of a benzophenone synthase (BPS), a type III polyketide synthase (PKS), was cloned and the recombinant protein expressed from the fruit pericarps of Garcinia mangostana L., which contains mainly prenylated xanthones. The obtained GmBPS showed an amino acid sequence identity of 77-78% with other plant BPSs belonging to the same family (Clusiaceae). The recombinant enzyme produced 2,4,6-trihydroxybenzophenone as the predominant product with benzoyl CoA as substrate. It also accepted other substrates, such as other plant PKSs, and used 1-3 molecules of malonyl CoA to form various phloroglucinol-type and polyketide lactone-type compounds. Thus, providing GmBPS with various substrates in vivo might redirect the xanthone biosynthetic pathway. PMID:22390826

  9. Formation of Helically Structured Chitin/CaCO3 Hybrids through an Approach Inspired by the Biomineralization Processes of Crustacean Cuticles.

    PubMed

    Matsumura, Shunichi; Kajiyama, Satoshi; Nishimura, Tatsuya; Kato, Takashi

    2015-10-01

    Chitin/CaCO3 hybrids with helical structures are formed through a biomineralization-inspired crystallization process under ambient conditions. Liquid-crystalline chitin whiskers are used as helically ordered templates. The liquid-crystalline structures are stabilized by acidic polymer networks which interact with the chitin templates. The crystallization of CaCO3 is conducted by soaking the templates in the colloidal suspension of amorphous CaCO3 (ACC) at room temperature. At the initial stage of crystallization, ACC particles are introduced inside the templates, and they crystallize to CaCO3 nanocrystals. The acidic polymer networks induce CaCO3 crystallization. The characterization of the resultant hybrids reveals that they possess helical order and homogeneous hybrid structures of chitin and CaCO3 , which resemble the structure and composition of the exoskeleton of crustaceans. PMID:26192070

  10. Hydroperoxide lyase and divinyl ether synthase.

    PubMed

    Grechkin, Alexander N

    2002-08-01

    "Heterolytic" hydroperoxide lyase (HPL) and divinyl ether synthase (DES) are important enzymes of the plant lipoxygenase pathway. HPL cleaves fatty acid hydroperoxides into the aldehyde fragments. DES converts hydroperoxides into the divinyl ethers. The present paper is concerned with recent studies on HPL and DES including their occurrence, properties, mechanisms of action, the cloning of their cDNAs and physiological importance of the enzymes and their products. PMID:12432936

  11. Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal?eosinophils

    PubMed Central

    Zanardo, Renata C. O.; Costa, Edmar; Ferreira, Heloisa H. A.; Antunes, Edson; Martins, Antonio R.; Murad, Ferid; De Nucci, Gilberto

    1997-01-01

    Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor N?-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10?8 M) and leukotriene B4 (LTB4, 10?8 M). l-NAME (but not d-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by l-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism. PMID:9391161

  12. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  13. Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

    PubMed

    Jones, Christopher G; Moniodis, Jessie; Zulak, Katherine G; Scaffidi, Adrian; Plummer, Julie A; Ghisalberti, Emilio L; Barbour, Elizabeth L; Bohlmann, Jörg

    2011-05-20

    Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of ?-, ?-, epi-?-santalene, and ?-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of ?-endo-bergamotene, ?-santalene, (Z)-?-farnesene, epi-?-santalene, and ?-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. PMID:21454632

  14. Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase

    PubMed Central

    Rising, Kathleen A.; Crenshaw, Charisse M.; Koo, Hyun Jo; Subramanian, Thangaiah; Chehade, Kareem A. H.; Starks, Courtney; Allen, Keith D.; Andres, Douglas A.; Spielmann, H. Peter; Noel, Joseph P.; Chappell, Joe

    2015-01-01

    As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids. PMID:25897591

  15. Infection Structure–Specific Expression of ?-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of ?-Glucan–Triggered Immunity in Maize[W

    PubMed Central

    Oliveira-Garcia, Ely; Deising, Holger B.

    2013-01-01

    ?-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of ?-1,3-glucan is unknown. We functionally characterized the ?-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive ?-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and ?-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of ?-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of ?-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while ?-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading ?-glucan–triggered immunity. PMID:23898035

  16. Proton pump inhibitors

    MedlinePLUS

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  17. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce (Pullman, WA); Burke, Charles Cullen (Moscow, ID)

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  18. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    PubMed Central

    2010-01-01

    Background We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Methods Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Results Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Conclusion Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco. PMID:20426864

  19. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  20. Divinyl ether synthase gene and protein, and uses thereof

    SciTech Connect

    Howe, Gregg A.; Itoh, Aya

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  1. Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide.

    PubMed

    Panaro, M A; Acquafredda, A; Lisi, S; Lofrumento, D D; Trotta, T; Satalino, R; Saccia, M; Mitolo, V; Brandonisio, O

    1999-01-01

    Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection. PMID:10592110

  2. Diacylglycerol Metabolism and Signaling is a Driving Force Underlying FASN Inhibitor Sensitivity in Cancer Cells

    PubMed Central

    Benjamin, Daniel I.; Li, Daniel S.; Lowe, Wallace; Heuer, Timothy; Kemble, George; Nomura, Daniel K.

    2015-01-01

    Fatty acid synthase (FASN) generates the de novo source of lipids for cell proliferation and is a promising cancer therapy target. Development of FASN inhibitors, however, necessitates a better understanding of sensitive and resistant cancer types to optimize patient treatment. Indeed, testing the cytotoxic effects of FASN inhibition across human cancer cells revealed diverse sensitivities. We show here that metabolic incorporation of glucose into specific complex lipid species strongly predicts FASN inhibitor sensitivity. We also show that the levels of one of these lipid classes, protein kinase C (PKC) stimulators diacylglycerols, are lowered upon FASN inhibitor treatment in sensitive compared to resistant cells and that PKC activators and inhibitors rescue cell death in sensitive cells and sensitize resistant cells, respectively. Our findings not only reveal a biomarker for predicting FASN sensitivity in cancer cells, but also a put forth a heretofore unrecognized mechanism underlying the anti-cancer effects of FASN inhibitors. PMID:25871544

  3. Glycinamide ribonucleotide synthase (EC 6.34.13)-aminoimidazole ribonucleotide synthase (EC 6.3.3.1) from Saccharomyces cerevisiae

    SciTech Connect

    Tret`yakov, O.Yu.; Ryzhova, T.A.; Smirnov, M.N.

    1995-12-01

    Here we report for the first time the isolation and properties of GAR synthase (E2)-AIR synthase (E5) (E2-E5) from the yeast Saccharomyces cerevisiae. A vector containing the S. cerevisiae ADE5,7 gene was used to obtain a yeast strain accumulating ADE5,7 coded E2-E5 in amounts equivalent to {approximately}20% of the total soluble protein. The pH optimum for E2 activity in forward reaction is 7.5, in the reverse reaction 7.5 - 8.5; the E5 activity optimum is at pH 8.0. The temperature optimum for E2 is 42{degrees}C (in the reverse reaction), 35{degrees}C for E5. Data on the pH and temperature stability of E2-E5 are presented. The E2 K{sub m} values are ({mu}M): 49 {+-}4 for glycine, 64 {+-}5 for PRA, 144 {+-} 10 for ATP, 21 {+-} 2 for GAR, 7 {+-} 1 for ADP, and 320 {+-} 30 for P{sub i}. The E5 K{sub m} values are ({mu}M): 80 {+-} 9 for GAM and 500 {+-} 52 for ATP. E2 and E5 are inactive in the absence of Mg{sup 2+}. E5 is K{sup +} -dependent. 2{prime}-dATP, GTP, ITP, UTP, and CTP are not substrates for E5, while AMP is a competitive E5 inhibitor relative to ATP (with K{sub i} = 700 {+-} 80 {mu}M). Hence, regulation of purine biosynthesis by one of its end products in S. cerevisiae occurs at the fifth stage of biosynthesis, supplementing the inhibition of the first enzyme. The molecular mass of the E2-E5 subunits is {approximately}87 kD. The native form is dimeric: the molecular masses of the enzyme determined by three different methods are 170, 175, and 200 kD. Short-term chymotrypsin treatment of E2-E5 cleaves the polypeptide chain, presumably in an interdomain region; as a result, E5 activity disappears while E2 activity is retained. 45 refs., 6 figs., 2 tabs.

  4. Physico-chemical characteristics and functional properties of chitin and chitosan produced by Mucor circinelloides using yam bean as substrate.

    PubMed

    Fai, Ana Elizabeth C; Stamford, Thayza C M; Stamford-Arnaud, Thatiana M; Santa-Cruz, Petrus D'Amorim; da Silva, Marta C Freitas; Campos-Takaki, Galba M; Stamford, Tânia L M

    2011-01-01

    Microbiological processes were used for chitin and chitosan production by Mucor circinelloides (UCP 050) grown in yam bean (Pachyrhizus erosus L. Urban) medium. The polysaccharides were extracted by alkali-acid treatment and structural investigations by X-ray diffraction, Fourier transform IR analysis, viscosity and thermal analysis by TG, DTG, and DTA were done. The highest biomass yield (20.7 g/L) was obtained at 96 hours. The highest levels of chitosan (64 mg/g) and chitin (500 mg/g) were produced at 48 and 72 hours, respectively. It was demonstrated that yam bean shows great potential as an economic medium and it is possible to achieve a good yield of chitosan with chemical properties that enable its use in biotechnological applications. PMID:21862956

  5. The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice.

    PubMed

    Miyata, Kana; Kozaki, Toshinori; Kouzai, Yusuke; Ozawa, Kenjirou; Ishii, Kazuo; Asamizu, Erika; Okabe, Yoshihiro; Umehara, Yosuke; Miyamoto, Ayano; Kobae, Yoshihiro; Akiyama, Kohki; Kaku, Hanae; Nishizawa, Yoko; Shibuya, Naoto; Nakagawa, Tomomi

    2014-11-01

    Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes. PMID:25231970

  6. Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation

    PubMed Central

    Baty, Ace M.; Eastburn, Callie C.; Diwu, Zhenjun; Techkarnjanaruk, Somkiet; Goodman, Amanda E.; Geesey, Gill G.

    2000-01-01

    The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97–N-acetyl-?-d-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase. PMID:10919822

  7. First evidence of chitin in calcified coralline algae: new insights into the calcification process of Clathromorphum compactum

    PubMed Central

    Rahman, M. Azizur; Halfar, Jochen

    2014-01-01

    Interest in calcifying coralline algae has been increasing over the past years due to the discovery of extensive coralline algal dominated ecosystems in Arctic and Subarctic latitudes, their projected sensitivity to ocean acidification and their utility as palaeoenvironmental proxies. Thus, it is crucial to obtain a detailed understanding of their calcification process. We here extracted calcified skeletal organic matrix components including soluble and insoluble fractions from the widely-distributed Subarctic and Arctic coralline alga Clathromorphum compactum. The lyophilized skeletal organic matrix fractions showed comparatively high concentrations of soluble and insoluble organic matrices comprising 0.9% and 4.5% of skeletal weight, respectively. This is significantly higher than in other skeletal marine calcifiers. Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) and X-Ray Diffraction (XRD) results indicate that chitin is present in the skeletal organic matrices of C. compactum. This polymer exhibits similar hierarchical structural organizations with collagen present in the matrix and serves as a template for nucleation and controls the location and orientation of mineral phases. Chitin contributes to significantly increasing skeletal strength, making C. compactum highly adapted for living in a shallow high-latitude benthic environment. Furthermore, chitin containing polysaccharides can increase resistance of calcifiers to negative effects of ocean acidification. PMID:25145331

  8. Fabrication and optimization of fast disintegrating tablets employing interpolymeric chitosan-alginate complex and chitin as novel superdisintegrants.

    PubMed

    Goel, Honey; Tiwary, Ashok K; Rana, Vikas

    2011-01-01

    The objective of the present work was to optimize the formulation of fast disintegrating tablets (FDTs) of ondansetron HCl containing novel superdisintegrants, possessing sufficient mechanical strength and disintegration time comparable to those containing crospovidone or croscarmellose sodium. The FDTs were formulated using a novel superdisintegrant (chitosan-alginate (1:1) interpolymer complex and chitin) to achieve a sweet tasting disintegrating system. The results revealed that chitin (5-20%) increased the porosity and decreased the DT of tablets. At higher concentrations chitin maintained tablet porosity even at 5.5 kg crushing strength. Ondansetron HCl was found to antagonize the wicking action of glycine. Further, evaluation of the mechanism of disintegration revealed that glycine transported the aqueous medium to different parts of the tablets while the chitosan-alginate complex swelled up due to transfer of moisture from glycine. This phenomenon resulted in breakage of the tablet within seconds. For preparing optimized FDTs, the reduced model equations generated from Box-Behnken design (BBD) were solved after substituting the known disintegration time of FDTs containing superdisintegrants in the reduced model equations. The results suggested that excipient system under investigation not only improved the disintegration time but also made it possible to prepare FDTs with higher crushing strength as compared to tablets containing known superdisintegrants. PMID:21796940

  9. Glycogen synthase kinase 3? regulates urine concentrating mechanism in mice.

    PubMed

    Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R; Kakade, Vijayakumar; Yu, Alan S L; Howard, Christiana; Rao, Reena

    2015-03-15

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3? and GSK3? isoforms. GSK3? has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3? has yet to be discerned. To investigate the role of GSK3? in urine concentration, we compared GSK3? knockout (GSK3?KO) mice with wild-type (WT) littermates. Under normal conditions, GSK3?KO mice had higher water intake and urine output. GSK3?KO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3?KO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3? in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3?KO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3? could play a crucial role in renal urine concentration and suggest that GSK3? might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus. PMID:25608967

  10. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 (?=?ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  11. Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

    PubMed Central

    Miyamoto, Katsushiro; Nukui, Eiji; Itoh, Hiroyuki; Sato, Takaji; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo; Inamori, Yoshihiko; Tsujibo, Hiroshi

    2002-01-01

    Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to ?-chitin and ?-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity. PMID:11889092

  12. Genetic variation in aldosterone synthase predicts plasma glucose levels

    E-print Network

    Botstein, David

    Genetic variation in aldosterone synthase predicts plasma glucose levels Koustubh Ranadeabc , Kwan single-nucleotide polymor- phisms in the aldosterone synthase gene and plasma glucose levels in a large disequilibrium in this population--were associated with fasting plasma glucose levels (P 0.000017) and those 60

  13. Orotic aciduria fibroblasts express a labile form of UMP synthase.

    PubMed

    Perry, M E; Jones, M E

    1989-09-15

    Orotic aciduria (type 1) results from a mutation in the gene for UMP synthase, a bifunctional protein containing the two enzyme activities which convert orotic acid and 5-phosphoribosyl-1-pyrophosphate to UMP and CO2. In fibroblasts from individuals with orotic aciduria, these two enzymatic activities are about 1% of normal but increase dramatically when deficient cells are grown in the presence of 6-azauridine. Using a polyclonal antiserum to sodium dodecyl sulfate-denatured, pure human UMP synthase, we show that fibroblasts from a patient with orotic aciduria have a low level of immunoreactive UMP synthase protein. Pulse-chase analysis reveals that the UMP synthase is degraded rapidly in the deficient cells. Growth of deficient cells in 6-azauridine leads to an increase in UMP synthase protein and its two enzymatic activities via a decreased rate of proteolytic degradation of UMP synthase. UMP synthase in extracts from deficient cells is more readily denatured by heat and is stabilized after growth of cells in 6-azauridine. These data suggest that the detrimental deficiency of this one patient results from a structurally altered UMP synthase that is probably present in low steady-state amounts due to proteolysis and that this labile protein can be stabilized against heat denaturation and proteolytic degradation by 6-aza-UMP. PMID:2475503

  14. Transient accumulation of elastic energy in proton translocating ATP synthase

    E-print Network

    Steinhoff, Heinz-Jürgen

    Hypothesis Transient accumulation of elastic energy in proton translocating ATP synthase Dmitry A 12 March 1999 Abstract ATP synthase is conceived as a rotatory engine with two reversible drives that the hydrolysis of three molecules of ATP in FI drives the shaft over a full circle in three steps of 120³ each

  15. ATP synthases from archaea: the beauty of a molecular motor.

    PubMed

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. PMID:24650628

  16. Genetics Home Reference: N-acetylglutamate synthase deficiency

    MedlinePLUS

    ... synthase deficiency is an inherited disorder that causes ammonia to accumulate in the blood. Ammonia, which is formed when proteins are broken down ... is especially sensitive to the effects of excess ammonia. N-acetylglutamate synthase deficiency may become evident in ...

  17. Glycogen synthase kinse-3 regulates endoplasmic reticulum (ER) stress-induced CHOP expression in neuronal cells

    PubMed Central

    Meares, Gordon P.; Mines, Marjelo A.; Beurel, Eleonore; Eom, Tae-Yeon; Song, Ling; Zmijewska, Anna A.; Jope, Richard S.

    2011-01-01

    Endoplasmic reticulum (ER) stress, often resulting from cellular accumulation of misfolded proteins, occurs in many neurodegenerative disorders, in part because of the relatively long lifetime of neurons. Excessive accumulation of misfolded proteins activates the unfolded protein response (UPR) that dampens protein synthesis and promotes removal of misfolded proteins to support survival of ER-stressed cells. However, the UPR also initiates apoptotic signaling to kill cells if recovery is not achieved. Thus, there is much interest in identifying determinants of the life-death switch and interventions that promote recovery and survival. One intervention that has consistently been shown to protect cells from ER stress-induced apoptosis is application of inhibitors of glycogen synthase kinase-3 (GSK3). Therefore, we examined where in the UPR pathway GSK3 inhibitors intercede to impede signaling towards apoptosis. Apoptosis following UPR activation can be mediated by activation of two transcription factors, ATF4 and ATF6, that activate expression of the death-inducing transcription factor C/EBP homologous protein (CHOP/GADD153) following ER stress. We found that ER stress induced activated ATF6 and ATF4, but these responses were not altered by pretreatment with GSK3 inhibitors. However, inhibition of GSK3 effectively reduced the expression of CHOP, and this was apparent in several types of neural-related cells and was evident after application of several structurally diverse GSK3 inhibitors. Therefore, reduction of CHOP activation provides one mechanism by which inhibitors of GSK3 are capable of shifting cell fate towards survival instead of apoptosis following ER stress. PMID:21356208

  18. Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker

    PubMed Central

    Liu, Hailan; Liu, Jing-Yuan; Wu, Xi; Zhang, Jian-Ting

    2010-01-01

    Human fatty acid synthase (FASN) is a 270-kDa cytosolic dimeric enzyme that is responsible for palmitate synthesis. FASN is slowly emerging and rediscovered as a marker for diagnosis and prognosis of human cancers. Recent studies showed that FASN is an oncogene and inhibition of FASN effectively and selectively kill cancer cells. With recent publications of the FASN crystal structure and the new development of FASN inhibitors, targeting FASN opens a new window of opportunity for metabolically combating cancers. In this article, we will review critically the recent progresses in understanding the structure, function, and the role of FASN in cancers and pharmacologically targeting FASN for human cancer treatment. PMID:20706604

  19. Suppression of fatty acid synthase, differentiation and lipid accumulation in adipocytes by curcumin.

    PubMed

    Zhao, Jiong; Sun, Xue-Bing; Ye, Fei; Tian, Wei-Xi

    2011-05-01

    Curcumin is a well-known component of the cook seasoning and traditional herb turmeric (Curcuma longa), which has been reported to prevent obesity. However, the mechanism still remains to be determined. In this study, curcumin is found to be an effective inhibitor of fatty acid synthase (FAS), and its effects on adipocytes are further evaluated. Curcumin shows both fast-binding and slow-binding inhibitions to FAS. Curcumin inhibits FAS with an IC?? value of 26.8 ?M, noncompetitively with respect to NADPH, and partially competitively against both substrates acetyl-CoA and malonyl-CoA. This suggests that the malonyl/acetyl transferase domain of FAS possibly is the main target of curcumin. The time-dependent inactivation shows that curcumin inactivates FAS with two-step irreversible inhibition, a specific reversible binding followed by an irreversible modification by curcumin. Like other classic FAS inhibitors, curcumin prevents the differentiation of 3T3-L1 cells, and thus represses lipid accumulation. In the meantime, curcumin decreases the expression of FAS, down-regulates the mRNA level of PPAR? and CD36 during adipocyte differentiation. Curcumin is reported here as a novel FAS inhibitor, and it suppresses adipocyte differentiation and lipid accumulation, which is associated with its inhibition of FAS. Hence, curcumin is considered to be having potential application in the prevention of obesity. PMID:21221723

  20. Identification and Characterization of an Allosteric Inhibitory Site on Dihydropteroate Synthase

    PubMed Central

    2015-01-01

    The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and quasi-harmonic analyses suggest that the effect of inhibitor binding is transmitted from the dimer interface to the active-site loops that are known to assume an obligatory ordered substructure during catalysis. Together with the kinetics results, these structural and dynamics data suggest an inhibitory mechanism in which binding at the dimer interface impacts loop movements that are required for product release. Our results potentially provide a novel target site for the development of new antibiotics. PMID:24650357

  1. Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. [Spinacia oleracea

    SciTech Connect

    Huber, J.L. ); Huber, S.C. North Carolina State Univ., Raleigh ); Hite, D.R.C.; Outlaw, W.H. Jr. )

    1991-01-01

    Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ({gamma}-{sup 32}P)ATP. Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25{degrees}C before assay. The spontaneous inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg{sup 2+} and was relatively insensitive to Ca{sup 2+} and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with ({sup 32}P)Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate.

  2. Activities and regulation of peptidoglycan synthases.

    PubMed

    Egan, Alexander J F; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-10-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN. PMID:26370943

  3. Activities and regulation of peptidoglycan synthases

    PubMed Central

    Egan, Alexander J. F.; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-01-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN. PMID:26370943

  4. Effects of Chitin and Sepia Ink Hybrid Hemostatic Sponge on the Blood Parameters of Mice

    PubMed Central

    Zhang, Wei; Sun, Yu-Lin; Chen, Dao-Hai

    2014-01-01

    Chitin and sepia ink hybrid hemostatic sponge (CTSH sponge), a new biomedical material, was extensively studied for its bene?cial biological properties of hemostasis and stimulation of healing. However, studies examining the safety of CTSH sponge in the blood system are lacking. This experiment aimed to examine whether CTSH sponge has negative effect on blood systems of mice, which were treated with a dosage of CTSH sponge (135 mg/kg) through a laparotomy. CTSH sponge was implanted into the abdominal subcutaneous and a laparotomy was used for blood sampling from abdominal aortic. Several kinds of blood parameters were detected at different time points, which were reflected by coagulation parameters including thrombin time (TT), prothrombin time (PT), activated partial thromboplatin time (APTT), fibrinogen (FIB) and platelet factor 4 (PF4); anticoagulation parameter including antithrombin III (AT-III); fibrinolytic parameters including plasminogen (PLG), fibrin degradation product (FDP) and D-dimer; hemorheology parameters including blood viscosity (BV) and plasma viscosity (PV). Results showed that CTSH sponge has no significant effect on the blood parameters of mice. The data suggested that CTSH sponge can be applied in the ?eld of biomedical materials and has potential possibility to be developed into clinical drugs of hemostatic agents. PMID:24727395

  5. A Novel Chitin Binding Crayfish Molar Tooth Protein with Elasticity Properties

    PubMed Central

    Tynyakov, Jenny; Bentov, Shmuel; Abehsera, Shai; Khalaila, Isam; Manor, Rivka; Katzir Abilevich, Lihie; Weil, Simy; Aflalo, Eliahu D.; Sagi, Amir

    2015-01-01

    The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties. PMID:26010981

  6. Photothermal and Structural Comparative Analysis of Chitinous Exoskeletons of Marine Invertebrates

    NASA Astrophysics Data System (ADS)

    Juárez-de la Rosa, B. A.; Yañez-Limón, J. M.; Tiburcio-Moreno, J. A.; Zambrano, M.; Ardisson, P.-L.; Quintana, P.; Alvarado-Gil, J. J.

    2012-11-01

    Chitinous materials are common in nature and provide different functions including protection and support of many invertebrate animals. Exoskeletons in these organisms constitute the boundary regulating interaction between the animal and the external environment. For this reason, it is important to study the physical properties of these skeletons, in particular, thermal properties. The objective of this study is to investigate the thermal diffusivity of the skeletons of four species of marine invertebrates, Antipathes caribbeana (black coral), Panulinus argus (lobster), Callinectes sapidus (crab), and Limulus polyphemus (xiphosure). Thermal characterization is performed using photothermal radiometry (PTR) and laser-flash techniques. The measurements are complemented with structural characterization using X-ray diffraction. The results using both laser flash and PTR are consistent. These indicate that the thermal properties are strongly dependent on the presence of biogenic minerals (calcium and/or magnesium) and on the crystallinity index of the structure. The thermal-diffusivity values show an increase as a function of the crystallinity index.

  7. Halo(natrono)archaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates

    PubMed Central

    Sorokin, Dimitry Y.; Toshchakov, Stepan V.; Kolganova, Tatyana V.; Kublanov, Ilya V.

    2015-01-01

    Until recently, extremely halophilic euryarchaeota were considered mostly as aerobic heterotrophs utilizing simple organic compounds as growth substrates. Almost nothing is known on the ability of these prokaryotes to utilize complex polysaccharides, such as cellulose, xylan, and chitin. Although few haloarchaeal cellulases and chitinases were recently characterized, the analysis of currently available haloarchaeal genomes deciphered numerous genes-encoding glycosidases of various families including endoglucanases and chitinases. However, all these haloarchaea were isolated and cultivated on simple substrates and their ability to grow on polysaccharides in situ or in vitro is unknown. This study examines several halo(natrono)archaeal strains from geographically distant hypersaline lakes for the ability to grow on insoluble polymers as a sole growth substrate in salt-saturated mineral media. Some of them belonged to known taxa, while other represented novel phylogenetic lineages within the class Halobacteria. All isolates produced extracellular extremely salt-tolerant cellulases or chitinases, either cell-free or cell-bound. Obtained results demonstrate a presence of diverse populations of haloarchaeal cellulo/chitinotrophs in hypersaline habitats indicating that euryarchaea participate in aerobic mineralization of recalcitrant organic polymers in salt-saturated environments. PMID:26441877

  8. Chitin-Lignin Material as a Novel Matrix for Enzyme Immobilization

    PubMed Central

    Zdarta, Jakub; Klapiszewski, ?ukasz; Wysokowski, Marcin; Norman, Ma?gorzata; Ko?odziejczak-Radzimska, Agnieszka; Moszy?ski, Dariusz; Ehrlich, Hermann; Maciejewski, Hieronim; Stelling, Allison L.; Jesionowski, Teofil

    2015-01-01

    Innovative materials were made via the combination of chitin and lignin, and the immobilization of lipase from Aspergillus niger. Analysis by techniques including FTIR, XPS and 13C CP MAS NMR confirmed the effective immobilization of the enzyme on the surface of the composite support. The electrokinetic properties of the resulting systems were also determined. Results obtained from elemental analysis and by the Bradford method enabled the determination of optimum parameters for the immobilization process. Based on the hydrolysis reaction of para-nitrophenyl palmitate, a determination was made of the catalytic activity, thermal and pH stability, and reusability. The systems with immobilized enzymes were found to have a hydrolytic activity of 5.72 mU, and increased thermal and pH stability compared with the native lipase. The products were also shown to retain approximately 80% of their initial catalytic activity, even after 20 reaction cycles. The immobilization process, using a cheap, non-toxic matrix of natural origin, leads to systems with potential applications in wastewater remediation processes and in biosensors. PMID:25903282

  9. Anti-inflammatory and antinociceptive activity of chitin-binding lectin from Canna limbata seeds.

    PubMed

    Araújo, Theolyta S; Teixeira, Claudener S; Falcão, Maria A P; Junior, Vanir R Pinto; Santiago, Mayara Quiroz; Benevides, Raquel G; Delatorre, Plínio; Martins, Jorge L; Alexandre-Moreira, Magna S; Cavada, Benildo S; Campesatto, Eliane A; Rocha, Bruno A M

    2013-12-01

    Lectins are a structurally heterogeneous group of proteins or glycoproteins with at least one noncatalytic domain binding reversibly to a specific mono- or oligosaccharide. Monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins. In this study, we evaluated anti-inflammatory and antinociceptive effects of monocot lectin from the Canna limbata seeds (CLL). To accomplish this, CLL was purified and subjected to pharmacological assays: abdominal writhing induced by acetic acid, formalin, hot plate and Zymosan A-induced peritonitis tests. The CLL was purified by chromatographic chitin column, and the relative mass of 21 kDa observed in electrophoresis was confirmed by electrospray mass spectrometry, which also revealed that purified CLL consists of a dimer having a weight of 49,676 Da. The CLL showed nociceptive activity in the acetic acid test as well as peripheral antinociceptive response. The CLL also showed anti-inflammatory effect with the reduction of inflammation in the formalin test and neutrophil migration into the peritoneal cavity. This is the first report of anti-inflammatory activity for a monocot lectin, and it suggests a new pharmacological tool to understand inflammatory and antinociceptive processes mediated through lectins. PMID:24013883

  10. A three-dimensionally chitin nanofiber/carbon nanotube hydrogel network for foldable conductive paper.

    PubMed

    Chen, Chuchu; Yang, Chuang; Li, Suiyi; Li, Dagang

    2015-12-10

    We reported a highly conductive nanocomposite made with multiwalled carbon nanotubes (MWCNTs) and chitin nanofibers (ChNFs). The MWCNTs were dispersed into ChNFs by the simple process of vacuum-filtration, forming a three-dimensional network structure. In this approach, MWCNT acted as a filler to introduce electron channel paths throughout the ChNF skeleton. And then, a hybrid hydrogel system (20 wt.% NaOH, -18 °C) was applied to prepare the ChNF/MWCNT gel-film followed with drying process. It is found that the resultant ChNF/MWCNT gel-film exposed much more MWCNT areas forming denser structure due to the shrinking of ChNFs after the gelation treatment. Compared with ChNF/MWCNT film, the one treated under hydrogel system (ChNF/MWCNT gel-film) exhibited almost twice higher conductivity (9.3S/cm for 50 wt.% MWCNTs in gel-film; whereas 4.7S/cm for 50 wt.% MWCNTs in film). Moreover, the facile and low-cost of this conductive paper may have great potential in development of foldable electronic devices. PMID:26428129

  11. Halo(natrono)archaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates.

    PubMed

    Sorokin, Dimitry Y; Toshchakov, Stepan V; Kolganova, Tatyana V; Kublanov, Ilya V

    2015-01-01

    Until recently, extremely halophilic euryarchaeota were considered mostly as aerobic heterotrophs utilizing simple organic compounds as growth substrates. Almost nothing is known on the ability of these prokaryotes to utilize complex polysaccharides, such as cellulose, xylan, and chitin. Although few haloarchaeal cellulases and chitinases were recently characterized, the analysis of currently available haloarchaeal genomes deciphered numerous genes-encoding glycosidases of various families including endoglucanases and chitinases. However, all these haloarchaea were isolated and cultivated on simple substrates and their ability to grow on polysaccharides in situ or in vitro is unknown. This study examines several halo(natrono)archaeal strains from geographically distant hypersaline lakes for the ability to grow on insoluble polymers as a sole growth substrate in salt-saturated mineral media. Some of them belonged to known taxa, while other represented novel phylogenetic lineages within the class Halobacteria. All isolates produced extracellular extremely salt-tolerant cellulases or chitinases, either cell-free or cell-bound. Obtained results demonstrate a presence of diverse populations of haloarchaeal cellulo/chitinotrophs in hypersaline habitats indicating that euryarchaea participate in aerobic mineralization of recalcitrant organic polymers in salt-saturated environments. PMID:26441877

  12. Effects of chitin and sepia ink hybrid hemostatic sponge on the blood parameters of mice.

    PubMed

    Zhang, Wei; Sun, Yu-Lin; Chen, Dao-Hai

    2014-04-01

    Chitin and sepia ink hybrid hemostatic sponge (CTSH sponge), a new biomedical material, was extensively studied for its bene?cial biological properties of hemostasis and stimulation of healing. However, studies examining the safety of CTSH sponge in the blood system are lacking. This experiment aimed to examine whether CTSH sponge has negative effect on blood systems of mice, which were treated with a dosage of CTSH sponge (135 mg/kg) through a laparotomy. CTSH sponge was implanted into the abdominal subcutaneous and a laparotomy was used for blood sampling from abdominal aortic. Several kinds of blood parameters were detected at different time points, which were reflected by coagulation parameters including thrombin time (TT), prothrombin time (PT), activated partial thromboplatin time (APTT), fibrinogen (FIB) and platelet factor 4 (PF4); anticoagulation parameter including antithrombin III (AT-III); fibrinolytic parameters including plasminogen (PLG), fibrin degradation product (FDP) and D-dimer; hemorheology parameters including blood viscosity (BV) and plasma viscosity (PV). Results showed that CTSH sponge has no significant effect on the blood parameters of mice. The data suggested that CTSH sponge can be applied in the ?eld of biomedical materials and has potential possibility to be developed into clinical drugs of hemostatic agents. PMID:24727395

  13. Effects of Surface-Deacetylated Chitin Nanofibers in an Experimental Model of Hypercholesterolemia

    PubMed Central

    Azuma, Kazuo; Nagae, Tomone; Nagai, Takeshi; Izawa, Hironori; Morimoto, Minoru; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Ito, Norihiko; Okamoto, Yoshiharu; Saimoto, Hiroyuki; Ifuku, Shinsuke

    2015-01-01

    This study evaluated the effects of oral administration of surface-deacetylated chitin nanofibers (SDACNFs) on hypercholesterolemia using an experimental model. All rats were fed a high cholesterol diet with 1% w/w cholesterol and 0.5% w/w cholic acid for 28 days. Rats were divided equally into four groups: the control group was administered 0.05% acetic acid dissolved in tap water, and the SDACNF, chitosan (CS), and cellulose nanofiber (CLNF) groups were administered 0.1% CNF, CS, or CLNF dissolved in the tap water, respectively, during the experimental period. Changes in body weight, intake of food and water, and organ weight were measured. Serum blood chemistry and histopathological examination of the liver were performed. Administration of SDACNF did not affect body weight change, food and water intake, or organ weights. Administration of SDACNF and CS decreased the diet-induced increase in serum total cholesterol, chylomicron, very-low-density lipoprotein, and phospholipid levels on day 14. Moreover, oral administration of SDACNFs suppressed the increase of alanine transaminase levels on day 29 and suppressed vacuolar degeneration and accumulation of lipid droplets in liver tissue. These data indicate that SDACNF has potential as a functional food for patients with hypercholesterolemia. PMID:26263969

  14. Effects of Surface-Deacetylated Chitin Nanofibers in an Experimental Model of Hypercholesterolemia.

    PubMed

    Azuma, Kazuo; Nagae, Tomone; Nagai, Takeshi; Izawa, Hironori; Morimoto, Minoru; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Ito, Norihiko; Okamoto, Yoshiharu; Saimoto, Hiroyuki; Ifuku, Shinsuke

    2015-01-01

    This study evaluated the effects of oral administration of surface-deacetylated chitin nanofibers (SDACNFs) on hypercholesterolemia using an experimental model. All rats were fed a high cholesterol diet with 1% w/w cholesterol and 0.5% w/w cholic acid for 28 days. Rats were divided equally into four groups: the control group was administered 0.05% acetic acid dissolved in tap water, and the SDACNF, chitosan (CS), and cellulose nanofiber (CLNF) groups were administered 0.1% CNF, CS, or CLNF dissolved in the tap water, respectively, during the experimental period. Changes in body weight, intake of food and water, and organ weight were measured. Serum blood chemistry and histopathological examination of the liver were performed. Administration of SDACNF did not affect body weight change, food and water intake, or organ weights. Administration of SDACNF and CS decreased the diet-induced increase in serum total cholesterol, chylomicron, very-low-density lipoprotein, and phospholipid levels on day 14. Moreover, oral administration of SDACNFs suppressed the increase of alanine transaminase levels on day 29 and suppressed vacuolar degeneration and accumulation of lipid droplets in liver tissue. These data indicate that SDACNF has potential as a functional food for patients with hypercholesterolemia. PMID:26263969

  15. The Correlation between Chitin and Acidic Mammalian Chitinase in Animal Models of Allergic Asthma

    PubMed Central

    Shen, Chia-Rui; Juang, Horng-Heng; Chen, Hui-Shan; Yang, Ching-Jen; Wu, Chia-Jen; Lee, Meng-Hua; Hwang, Yih-Shiou; Kuo, Ming-Ling; Chen, Ya-Shan; Chen, Jeen-Kuan; Liu, Chao-Lin

    2015-01-01

    Asthma is the result of chronic inflammation of the airways which subsequently results in airway hyper-responsiveness and airflow obstruction. It has been shown that an elicited expression of acidic mammalian chitinase (AMCase) may be involved in the pathogenesis of asthma. Our recent study has demonstrated that the specific suppression of elevated AMCase leads to reduced eosinophilia and Th2-mediated immune responses in an ovalbumin (OVA)-sensitized mouse model of allergic asthma. In the current study, we show that the elicited expression of AMCase in the lung tissues of both ovalbumin- and Der P2-induced allergic asthma mouse models. The effects of allergic mediated molecules on AMCase expression were evaluated by utilizing promoter assay in the lung cells. In fact, the exposure of chitin, a polymerized sugar and the fundamental component of the major allergen mite and several of the inflammatory mediators, showed significant enhancement on AMCase expression. Such obtained results contribute to the basis of developing a promising therapeutic strategy for asthma by silencing AMCase expression. PMID:26580611

  16. Influence of interfacial interactions on deformation mechanism and interface viscosity in ?-chitin-calcite interfaces.

    PubMed

    Qu, Tao; Verma, Devendra; Alucozai, Milad; Tomar, Vikas

    2015-10-01

    The interfaces between organic and inorganic phases in natural materials have a significant effect on their mechanical properties. This work presents a quantification of the interface stress as a function of interface chemical changes (water, organic molecules) in chitin-calcite (CHI-CAL) interfaces using classical non-equilibrium molecular dynamics (NEMD) simulations and steered molecular dynamics (SMD) simulations. NEMD is used to investigate interface stress as a function of applied strain based on the virial stress formulation. SMD is used to understand interface separation mechanism and to calculate interfacial shear stress based on a viscoplastic interfacial sliding model. Analyses indicate that interfacial shear stress combined with shear viscosity can result in variations to the mechanical properties of the examined interfacial material systems. It is further verified with Kelvin-Voigt and Maxwell viscoelastic analytical models representing viscous interfaces and outer matrix. Further analyses show that overall mechanical deformation depends on maximization of interface shear strength in such materials. This work establishes lower and upper bounds of interface strength in the interfaces examined. PMID:26143601

  17. Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump.

    PubMed

    Laubinger, W; Dimroth, P

    1988-09-20

    The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter. PMID:2905167

  18. Para-Aminobenzoic Acid (PABA) Synthase Enhances Thermotolerance of Mushroom Agaricus bisporus

    PubMed Central

    Lu, Zhonglei; Kong, Xiangxiang; Lu, Zhaoming; Xiao, Meixiang; Chen, Meiyuan; Zhu, Liang; Shen, Yuemao; Hu, Xiangyang; Song, Siyang

    2014-01-01

    Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom’s innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins. PMID:24614118