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Sample records for intracellular pteroylpolyglutamate hydrolase

  1. Further characterization of intestinal lactase/phlorizin hydrolase

    DEFF Research Database (Denmark)

    Skovbjerg, H; Norén, O; Sjöström, H

    1982-01-01

    Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the pres......Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis...... in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig...... membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase....

  2. Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity

    NARCIS (Netherlands)

    Goosen, C.; Yuan, X.L.; Munster, J.M. van; Ram, A.F.J.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2007-01-01

    A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger.

  3. Glycoside hydrolases having multiple hydrolase activities

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

    2017-08-08

    Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

  4. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  5. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    OpenAIRE

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

  6. Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

    2007-01-01

    The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors

  7. Variants of glycoside hydrolases

    Science.gov (United States)

    Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  8. Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

    OpenAIRE

    Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

    2003-01-01

    A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna,...

  9. Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

    Science.gov (United States)

    Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

    2003-03-01

    A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

  10. The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Klein, Isabella; Korber, Martina; Athenstaedt, Karin; Daum, Günther

    2017-12-01

    In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  12. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Wu Jiajie

    2010-10-01

    Full Text Available Abstract Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice, the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar. To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model and Sorghum bicolor (sorghum. We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51, we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets

  13. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    Science.gov (United States)

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

  14. Use of full recovery hydrolasing equipment for facility decommissioning - 16325

    International Nuclear Information System (INIS)

    Martin, Scott A.; Adams, Scott R.

    2009-01-01

    The removal of surface contamination is a major challenge for nearly all nuclear facilities undergoing, or awaiting, decommissioning. Conventional means of surface decontamination can expose workers to unnecessary hazards, and are often not fit-for-purpose due to size constraints or weight restrictions. Additionally, conventional methods are not always easily deployed remotely due to their complexity or required services. The use of ultra high pressure water for surface decontamination, known as hydrolasing, is recognized as a technology which can be used in various applications requiring surface removal. Hydrolasing is an advantageous technology for many reasons including its versatility, overall simplicity and relative ease of remote deployment. For the nuclear industry, one of the largest challenges with regards to the use of hydrolasing is the requirement for the full recovery of the injected water and removed solids. For nonnuclear applications, there is often no requirement for recovery of the liquid and solid waste, which has led to few system designs which will recover the waste in full. S.A. Robotics' experience with the deployment of ultra high pressure water systems for nuclear applications has shown that full recovery of injected water and removed solids is achievable in both underwater and in-air applications. Innovative equipment and system design have allowed S.A. Robotics' hydrolasing systems to achieve near 100% solid and liquid recovery during concrete hydrolasing. This technology has been deployed for Fluor Hanford at Hanford's K-Basins, as well as for UKAEA as part of the Windscale Piles decommissioning project. The purpose of this paper is to provide a short description of the hydrolasing process and the associated waste issues, describe the unique design features of S.A. Robotics' hydrolasing systems which combat these issues, and provide an overview of two of the hydrolasing projects that S.A. Robotics has completed. (authors)

  15. ATPase and GTPase Tangos Drive Intracellular Protein Transport.

    Science.gov (United States)

    Shan, Shu-Ou

    2016-12-01

    The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  17. Pain and beyond: fatty acid amides and fatty acid amide hydrolase inhibitors in cardiovascular and metabolic diseases.

    Science.gov (United States)

    Pillarisetti, Sivaram; Alexander, Christopher W; Khanna, Ish

    2009-12-01

    Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of several important endogenous fatty acid amides (FAAs), including anandamide, oleoylethanolamide and palmitoylethanolamide. Because specific FAAs interact with cannabinoid and vanilloid receptors, they are often referred to as 'endocannabinoids' or 'endovanilloids'. Initial interest in this area, therefore, has focused on developing FAAH inhibitors to augment the actions of FAAs and reduce pain. However, recent literature has shown that these FAAs - through interactions with unique receptors (extracellular and intracellular) - can induce a diverse array of effects that include appetite suppression, modulation of lipid and glucose metabolism, vasodilation, cardiac function and inflammation. This review gives an overview of FAAs and diverse FAAH inhibitors and their potential therapeutic utility in pain and non-pain indications.

  18. Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

    Directory of Open Access Journals (Sweden)

    Olga Senko

    2017-12-01

    Full Text Available We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His6-OPH in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His6-OPH: increased levels of the total OPH-activity (up to 37% were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold use of PFH for biosynthesis of intracellular recombinant protein His6-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP, is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

  19. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  20. Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

    Science.gov (United States)

    Cibik, R; Chapot-Chartier, M P

    2000-11-01

    The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

  1. Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

    Science.gov (United States)

    Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

    2006-09-01

    A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

  2. Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

    Directory of Open Access Journals (Sweden)

    Fickers P.

    2008-01-01

    Full Text Available ipases are atypical hydrolases: principal characteristics and applications. Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field.

  3. In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

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    Christina Hung Hung Ha

    2015-01-01

    Full Text Available Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol. These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases.

  4. Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

    Directory of Open Access Journals (Sweden)

    Serik Shaikhin

    2014-01-01

    Full Text Available Introduction: Peptidoglycan (PG is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation. Methods. Lactococcus lactis 17А and Lactococcus garvieae 19А were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity. Results. According to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity. Conclusions. Use of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

  5. How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

    Directory of Open Access Journals (Sweden)

    Barth Sandra

    2005-04-01

    Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

  6. Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

    Science.gov (United States)

    Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

    2017-09-02

    The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Crystallization of mouse S-adenosyl-l-homocysteine hydrolase

    International Nuclear Information System (INIS)

    Ishihara, Masaaki; Kusakabe, Yoshio; Ohsumichi, Tsuyoshi; Tanaka, Nobutada; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

    2010-01-01

    Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation. S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress

  8. The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

    DEFF Research Database (Denmark)

    Champion, Elise; Remaud-Simeon, Magali; Skov, Lars Kobberøe

    2009-01-01

    Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris...... of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants...

  9. Hydrolase activity in Jerusalem artichoke and chicory

    Energy Technology Data Exchange (ETDEWEB)

    Klaushofer, H.; Abraham, B.; Leichtfried, G.

    1988-03-01

    Post-harvest storage of chicory and Jerusalem artichoke and overwintering of Jerusalem artichoke in the soil cause a more or less pronounced shortening of the fructan chain, depending on the variety. The proportion of fructose in the total fructan thus shifts towards glucose. This reduction on the fructose/glucose ratio is undesirable if the intention is to obtain a sweetener of high fructose content. In this work an attempt was made, via the quantity of fructose formed after a 4(3)-hour reaction of a tuber (root) extract with inulin, to assign a characteristic value to the depolymerization tendency of the material in question. However, since the plant extract not only contains enzymes (hydrolase A and B) that shorten the fructan chains but the activity of fructosyltransferase (SST, FFT) and enzymes of microbial origin (inulinase II, invertase) must also be considered, the concept of 'hydrolase activity' used by the authors is essentially an expression of 'total activity'. The activity unit (EU) is defined as the ability to split of 1 ..mu..mol of fructose from (chicory) inulin per minute under experimental conditions. Values of 0.25 to 0.77 EU/g dry solids were found in Jerusalem artichoke. Considerable differences may occur between varieties from the same cultivated area and the same harvest period. With one and the same variety, the activity appears to be subject to marked yearly fluctuations, so that at present, because of hydrolase activity, nothing certain can be said about the depolymerization tendency of a variety.

  10. Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; DeVeaux, Linda C.

    2018-04-16

    Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader

  11. A remote but significant sequence homology between glycoside hydrolase clan GH-H and glycoside hydrolase family GH 31

    DEFF Research Database (Denmark)

    Janecek, S.; Svensson, Birte; MacGregor, E.A.

    2007-01-01

    Although both the α-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is...

  12. HYDROLASING OF CONTAMINATED UNDERWATER BASIN SURFACES AT THE HANFORD K AREA

    International Nuclear Information System (INIS)

    CHRONISTER, G.B.

    2005-01-01

    This paper discusses selecting and implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water. sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal

  13. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2014-01-01

    of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α...... investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases....

  14. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Science.gov (United States)

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  15. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Su

    Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  16. Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wenxin; Wang, Qihai; Bi, Ruchang, E-mail: rcbi@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2005-12-01

    The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap{sub 4}A) hydrolase (EC 3.6.1.41) hydrolyzes Ap{sub 4}A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap{sub 4}A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap{sub 4}A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2{sub 1}, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°.

  17. Generation and characterization of epoxide hydrolase 3 (EPHX3-deficient mice.

    Directory of Open Access Journals (Sweden)

    Samantha L Hoopes

    Full Text Available Cytochrome P450 (CYP epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs, which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH and soluble epoxide hydrolase (EPHX2, sEH were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT. Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic

  18. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Břicháč, Jiří; Kyslík, Pavel

    2005-01-01

    Roč. 120, - (2005), s. 364-375 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z5020903 Keywords : screening * epoxide hydrolase * biotransformation Subject RIV: EE - Microbiology, Virology Impact factor: 2.687, year: 2005

  19. ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

    Science.gov (United States)

    Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R; Kim, Chung Sub; Herzon, Seth B; Bruner, Steven D; Crawford, Jason M

    2017-12-13

    Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

  20. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    International Nuclear Information System (INIS)

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  1. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil [Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States); Servinsky, Matthew D. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Gerlach, Elliot S. [Federal Staffing Resources, 2200 Somerville Road, Annapolis, MD 21401 (United States); Sund, Christian J. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Hurley, Margaret M., E-mail: katherine.germane.civ@mail.mil [US Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD 21005 (United States); Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States)

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  2. Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

    NARCIS (Netherlands)

    Zandleven, J.S.

    2006-01-01

    Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food

  3. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

    1998-01-01

    The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

  4. Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

    NARCIS (Netherlands)

    Ariës-Kronenburg, N.A.E.

    2002-01-01

    Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like

  5. Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence.

    Directory of Open Access Journals (Sweden)

    Ping Wang

    Full Text Available Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

  6. Cytosolic cholesterol ester hydrolase in adrenal cortex

    OpenAIRE

    Tocher, Douglas R.

    1983-01-01

    Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

  7. A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

    Science.gov (United States)

    Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

  8. Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

    International Nuclear Information System (INIS)

    Buchko, Garry W.; Edwards, Thomas E.; Abendroth, Jan; Arakaki, Tracy L.; Law, Laura; Napuli, Alberto J.; Hewitt, Stephen N.; Van Voorhis, Wesley C.; Stewart, Lance J.; Staker, Bart L.; Myler, Peter J.

    2011-01-01

    B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution. Cat scratch fever (also known as cat scratch disease and bartonellosis) is an infectious disease caused by the proteobacterium Bartonella henselae following a cat scratch. Although the infection usually resolves spontaneously without treatment in healthy adults, bartonellosis may lead to severe complications in young children and immunocompromised patients, and there is new evidence suggesting that B. henselae may be associated with a broader range of clinical symptoms then previously believed. The genome of B. henselae contains genes for two putative Nudix hydrolases, BH02020 and BH01640 (KEGG). Nudix proteins play an important role in regulating the intracellular concentration of nucleotide cofactors and signaling molecules. The amino-acid sequence of BH02020 is similar to that of the prototypical member of the Nudix superfamily, Escherichia coli MutT, a protein that is best known for its ability to neutralize the promutagenic compound 7,8-dihydro-8-oxoguanosine triphosphate. Here, the crystal structure of BH02020 (Bh-MutT) in the Mg 2+ -bound state was determined at 2.1 Å resolution. As observed in all Nudix hydrolase structures, the α-helix of the highly conserved ‘Nudix box’ in Bh-MutT is one of two helices that sandwich a four-stranded mixed β-sheet with the central two β-strands parallel to each other. The catalytically essential divalent cation observed in the Bh-MutT structure, Mg 2+ , is coordinated to the side chains of Glu57 and Glu61. The structure is not especially robust; a temperature melt obtained using circular dichroism spectroscopy shows that Bh-MutT irreversibly unfolds and precipitates out of solution upon heating, with a T m of 333 K

  9. Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

    Science.gov (United States)

    Bera, Asim K; Aukema, Kelly G; Elias, Mikael; Wackett, Lawrence P

    2017-03-27

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

  10. Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

    Science.gov (United States)

    Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

    2011-01-01

    Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

  11. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    Science.gov (United States)

    1986-07-01

    M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...did get re-adjusted after the sample was applied. After the sample was applied the column was washed with the above MES buffer an.+eluted with 100 ml...Lieske (94) and compared them to the reversed phase HPLC retention times we have previously reported (16). We get an excellent linear correlation

  12. Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

    Science.gov (United States)

    Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

    2016-02-01

    Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

    Science.gov (United States)

    Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

    2015-01-14

    Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

  14. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    Energy Technology Data Exchange (ETDEWEB)

    Khristov, D; Marinopolski, G

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions.

  15. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    International Nuclear Information System (INIS)

    Khristov, D.; Marinopolski, G.

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions

  16. Structural insight into catalytic mechanism of PET hydrolase

    OpenAIRE

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-01-01

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  17. Structural insight into catalytic mechanism of PET hydrolase.

    Science.gov (United States)

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-12-13

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  18. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

    NARCIS (Netherlands)

    Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

    2003-01-01

    A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

  19. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2017-12-26

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

  20. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-03-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

  1. A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography.

    Science.gov (United States)

    Park, Chang-Su; Yang, Hee-Jong; Kim, Dong-Ho; Kang, Dae-Ook; Kim, Min-Soo; Choi, Nack-Shick

    2012-06-01

    A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

  2. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.; Yang, J.; Morisseau, C.; German, J. B.; Scott-Van Zeeland, A. A.; Armando, A. M.; Quehenberger, O.; Bergen, A. W.; Magistretti, Pierre J.; Berrettini, W.; Halmi, K. A.; Schork, N.; Hammock, B. D.; Kaye, W.

    2015-01-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product

  3. High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

    2017-01-01

    A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

  4. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes : Prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    NARCIS (Netherlands)

    Jiménez Avella, Diego; Dini Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered

  5. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    International Nuclear Information System (INIS)

    Pavlidis, Ioannis V.; Vorhaben, Torge; Gournis, Dimitrios; Papadopoulos, George K.; Bornscheuer, Uwe T.; Stamatis, Haralambos

    2012-01-01

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme–nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme–nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  6. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Pavlidis, Ioannis V. [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece); Vorhaben, Torge [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Gournis, Dimitrios [University of Ioannina, Department of Materials Science and Engineering (Greece); Papadopoulos, George K. [Epirus Institute of Technology, Laboratory of Biochemistry and Biophysics, Faculty of Agricultural Technology (Greece); Bornscheuer, Uwe T. [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Stamatis, Haralambos, E-mail: hstamati@cc.uoi.gr [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece)

    2012-05-15

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme-nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme-nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  7. S-Inosyl-L-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-L-Methionine Recycling.

    Science.gov (United States)

    Miller, Danielle; Xu, Huimin; White, Robert H

    2015-07-01

    S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD(+) and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the

  8. IMMOBILIZATION OF TANNIN ACYL HYDROLASE FROM ASPERGILLUS NIGER

    OpenAIRE

    B. Lenin Kumar*, N. Lokeswari and D. Sriramireddy

    2013-01-01

    ABSTRACT: Tannin acyl hydrolase, commonly referred to as tannase (E.C. 3.1.1.20), an inducible extra-cellular enzyme produced by a number of animals, plants and microbes. In this investigation, tannase production under solid-state fermentation by using Aspergillus niger and the waste residue of cashew husk was used as substrate for obtaining the desired fermented product. Microbial tannase is more stable than tannase from other sources like plants or animals. Tannase from fungal sources are r...

  9. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  10. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Directory of Open Access Journals (Sweden)

    María Inés Marchesini

    Full Text Available Choloylglycine hydrolase (CGH, E.C. 3.5.1.24 is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  11. Listeria monocytogenes PdeE, a phosphodiesterase that contributes to virulence and has hydrolytic activity against cyclic mononucleotides and cyclic dinucleotides

    Science.gov (United States)

    We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365'2464, to adhere...

  12. Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides

    NARCIS (Netherlands)

    Zandleven, J.S.; Beldman, G.; Bosveld, M.; Benen, J.A.E.; Voragen, A.G.J.

    2005-01-01

    XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of ¿-D-galacturonic acid, highly substituted with ß-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were

  13. Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

    NARCIS (Netherlands)

    Kronenburg, N.A.E.; Bont, de J.A.M.; Fischer, L.

    2001-01-01

    The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were

  14. Enzymatic degradation studies of xylogalacturonans from apple and potato, using xylogalacturonan hydrolase

    NARCIS (Netherlands)

    Zandleven, J.S.; Beldman, G.; Bosveld, M.; Schols, H.A.; Voragen, A.G.J.

    2006-01-01

    Action of xylogalacturonan hydrolase (XGH) towards xylogalacturonan (XGA) present in the alkali saponified ¿modified hairy regions¿ from potato and apple pectin was studied. Analysis of enzymatic degradation products from XGA in these complex pectins demonstrated that the degradable

  15. Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

    International Nuclear Information System (INIS)

    Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

    2008-01-01

    T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

  16. Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

    Science.gov (United States)

    Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

    2016-02-01

    The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

  17. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    International Nuclear Information System (INIS)

    Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

    1986-01-01

    The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6- 3 H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6- 3 H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells

  18. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer...

  19. Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Dandanell, Gert

    2005-01-01

    as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 k...... the neutral form of xanthosine....

  20. Evaluation of fish models of soluble epoxide hydrolase inhibition.

    OpenAIRE

    Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

    2001-01-01

    Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrol...

  1. Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Kyslík, Pavel

    2006-01-01

    Roč. 1760, - (2006), s. 245-252 ISSN 0006-3002 Institutional research plan: CEZ:AV0Z50200510 Keywords : epoxide hydrolase * enantioselectivity * aspergillus niger Subject RIV: EE - Microbiology, Virology

  2. Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

    Directory of Open Access Journals (Sweden)

    Juliane Schmidt

    2017-02-01

    Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

  3. Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii.

    Directory of Open Access Journals (Sweden)

    Riccardo Arrigucci

    Full Text Available Bacterial cell division ends with the separation of the daughter cells, a process that requires peptidoglycan hydrolases (PGHs. Bacteria lacking cell separating PGHs are impaired in cell separation with the formation of long chains or clusters. We identified a gene in Streptococcus gordonii encoding for a putative glucosaminidase (lytB. The lytB isogenic mutant grew in long bacterial chains and resulted in impaired biofilm formation. Purified recombinant LytB showed a murolytic activity on Micrococcus lysodeikticus cell suspension and was able to disperse the long chains of the mutant, restoring the wild type diplococci/short chain phenotype. LytB protein was localized only in culture supernatant cell fraction of S. gordonii, and co-cultures of wild type and lytB mutant showed a significant reduction of bacterial chain length, indicating that LytB is a secreted enzyme. Our results demonstrate that LytB is a secreted peptidoglycan hydrolase required for S. gordonii cell separation.

  4. Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family.

    Science.gov (United States)

    Zhang, L; Godzik, A; Skolnick, J; Fetrow, J S

    1998-01-01

    Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures. The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family. The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.

  5. Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog.

    Science.gov (United States)

    Singh, Neha; Dalal, Vikram; Mahto, Jai Krishna; Kumar, Pravindra

    2017-09-15

    Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%-84.4% of DMP, 75.0%-75.7% of DEP and 71.7%-74.7% of DEHP, initial amount of each phthalate is 500mgL -1 of each phthalate, after 44h of incubation. GC-MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

    Danielsen, E M

    1990-01-01

    of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic......The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal...... explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation...

  7. Transient changes of enzyme activity of five acid hydrolases in the supernatants of homogenates of hearts of mice due to ultraviolet irradiation

    International Nuclear Information System (INIS)

    Droba, B.; Jagiellonian Univ., Krakow

    1977-01-01

    Enzymatic activity of five lysosomal hydrolases: acid p-nitrophenyl phosphatase (EC 3.1.3.2), acid β-glycerophosphatase (EC 3.1.3.2), arylsulphatase (EC 3.1.6.1), β-galactosidase (EC 3.2.1.23) and β-N-acetylhexoaminidase (EC 3.2.1.30) was studied in the supernatants of homogenates of hearts of unirradiated mice, serving as controls, and a group of UV-irradiated mice. In the control group, determinations made at 6-hr intervals showed rhythmic diurnal changes in activities of three acid hydrolases. These changes were statistically significant in the case of acid p-nitrophenyl phosphatase, acid β-glycerophosphatase, and β-N-acetylhexosaminidase. The effect of UV-irradiation was manifested mainly by depression of enzyme activities of the acid hydrolases during the first few hours after exposure. Depression of activities of arylsulphatase and β-N-acetylhexosaminidase by UV light was statistically significant. Presumably, the fall in enzyme activities of the acid hydrolases was due to chemical mediators formed in the skin under the influence of UV-radiation and adrenal corticoids secreted into the blood

  8. The role of epoxide hydrolase Y113H gene variant in pancreatic diseases.

    NARCIS (Netherlands)

    Ockenga, J.; Strunck, S.; Post, C.; Schulz, H.U.; Halangk, J.; Pfutzer, R.H.; Lohr, M.; Oettle, H.; Kage, A.; Rosendahl, J.; Keim, V.; Drenth, J.P.H.; Jansen, J.B.M.J.; Lochs, H.; Witt, H.

    2009-01-01

    OBJECTIVES: Chronic pancreatitis (CP) and pancreatic adenocarcinoma (pCA) are associated with risk factors such as alcohol intake and tobacco smoking. Microsomal epoxide hydrolase (EPHX1) is a phase II detoxifying enzyme capable of tobacco-borne toxicant inactivation. We studied the role of the

  9. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    Directory of Open Access Journals (Sweden)

    Luis V. Rodríguez-Durán

    2011-01-01

    Full Text Available Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.

  10. Novel strategies for upstream and downstream processing of tannin acyl hydrolase.

    Science.gov (United States)

    Rodríguez-Durán, Luis V; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.

  11. Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

    NARCIS (Netherlands)

    Visser, H.; Vreugdenhil, S.; Bont, de J.A.M.; Verdoes, J.C.

    2000-01-01

    We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of

  12. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases

    DEFF Research Database (Denmark)

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav

    2016-01-01

    , the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities....

  13. Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

    Science.gov (United States)

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

    2016-01-01

    Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

  14. Crystal structure of bile salt hydrolase from Lactobacillus salivarius.

    Science.gov (United States)

    Xu, Fuzhou; Guo, Fangfang; Hu, Xiao Jian; Lin, Jun

    2016-05-01

    Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 Å resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 Å (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity.

  15. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    Energy Technology Data Exchange (ETDEWEB)

    Brzezinski, Krzysztof [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland); Bujacz, Grzegorz [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Faculty of Food Chemistry and Biotechnology, Technical University of Lodz (Poland); Jaskolski, Mariusz, E-mail: mariuszj@amu.edu.pl [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland)

    2008-07-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.

  16. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    International Nuclear Information System (INIS)

    Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-01-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4 3 2 1 2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4 3 2 1 2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation

  17. Glycoside Hydrolases across Environmental Microbial Communities.

    Directory of Open Access Journals (Sweden)

    Renaud Berlemont

    2016-12-01

    Full Text Available Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities.

  18. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    International Nuclear Information System (INIS)

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-01-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC 50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined

  19. Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

    DEFF Research Database (Denmark)

    Holt, S.; J. Fowler, C.; Rocksén, D.

    2004-01-01

    The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor a into the bronchoalveolar......-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent...... inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity....

  20. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

    Science.gov (United States)

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-07-01

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

  1. Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

    Science.gov (United States)

    Long, Aaron; Klimova, Nina; Kristian, Tibor

    2017-10-01

    NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

  2. Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression

    NARCIS (Netherlands)

    Verhave, Menno; Krasinski, Stephen D.; Christian, Sara I.; van Schaik, Sandrijn; van den Brink, Gijs R.; Doting, Edwina M. H.; Maas, Saskia M.; Wolthers, Katja C.; Grand, Richard J.; Montgomery, Robert K.

    2004-01-01

    OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb

  3. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    Energy Technology Data Exchange (ETDEWEB)

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  4. In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2015-01-01

    Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

  5. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chao-Yu; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Chou, Chia-Cheng; Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

    2006-06-01

    The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

  6. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  7. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  8. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    Science.gov (United States)

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-03

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. The Serine Hydrolase ABHD6 Is a Critical Regulator of the Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Gwynneth Thomas

    2013-10-01

    Full Text Available The serine hydrolase α/β hydrolase domain 6 (ABHD6 has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

  10. α/β-Hydrolase Domain 6 in the Ventromedial Hypothalamus Controls Energy Metabolism Flexibility

    Directory of Open Access Journals (Sweden)

    Alexandre Fisette

    2016-10-01

    Full Text Available α/β-Hydrolase domain 6 (ABHD6 is a monoacylglycerol hydrolase that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG. Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMHKO have higher VMH 2-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMHKO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet. Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility.

  11. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  12. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    Science.gov (United States)

    Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

    2014-07-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

  13. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

    Energy Technology Data Exchange (ETDEWEB)

    Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D' haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

    2009-11-15

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  14. Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

    Science.gov (United States)

    Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

    2018-03-12

    Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

    2011-05-11

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  16. Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

    Science.gov (United States)

    Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

    2013-09-13

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

  17. Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

    Science.gov (United States)

    Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

    2013-01-01

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

  18. Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties

    OpenAIRE

    Astarita, G; Di Giacomo, B; Gaetani, S; Oveisi, F; Compton, TR; Rivara, S; Tarzia, G; Mor, M; Piomelli, D

    2006-01-01

    Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-α (PPAR-α). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-α with high potency in...

  19. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol...

  20. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  1. Screening brazilian macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases

    OpenAIRE

    Schinke, Cláudia; Germani, Jose Carlos

    2012-01-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipas...

  2. Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

    NARCIS (Netherlands)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical

  3. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  4. Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

    Science.gov (United States)

    Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

    2015-07-28

    Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We

  5. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community.

    Directory of Open Access Journals (Sweden)

    Martin Allgaier

    Full Text Available Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  6. AMPEROMETRIC THICK-FILM STRIP ELECTRODES FOR MONITORING ORGANOPHOSPHATE NERVE AGENTS BASED ON IMMOBILIZED ORGANOPHOSPHORUS HYDROLASE. (R823663)

    Science.gov (United States)

    An amperometric biosensor based on the immobilization of organophosphorus hydrolase(OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-costdetection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and ra...

  7. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    Science.gov (United States)

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  8. Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, van der C.J.B.; Werf, van der M.J.

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  9. Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, C.J.B. van der; Werf, M.J. van der

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  10. Acetobacter turbidans α-Amino Acid Ester Hydrolase. How a Single Mutation Improves an Antibiotic-Producing Enzyme

    NARCIS (Netherlands)

    Barends, Thomas R.M.; Polderman-Tijmes, Jolanda J.; Jekel, Peter A.; Williams, Christopher; Wybenga, Gjalt; Janssen, Dick B.; Dijkstra, Bauke W.

    2006-01-01

    The α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as

  11. Variation in bleomycin hydrolase gene is associated with reduced survival after chemotherapy for testicular germ cell cancer

    NARCIS (Netherlands)

    de Haas, Esther C.; Zwart, Nynke; Meijer, Coby; Nuver, Janine; Boezen, H. Marike; Suurmeijer, Albert J. H.; Hoekstra, Harald J.; van der Steege, Gerrit; Sleijfer, Dirk Th.; Gietema, Jourik A.

    2008-01-01

    Purpose Response to chemotherapy may be determined by gene polymorphisms involved in metabolism of cytotoxic drugs. A plausible candidate is the gene for bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, an essential component of chemotherapy regimens for disseminated testicular

  12. Optimization of the fermentation conditions and substrate specifity of mycelium-bound ester hydrolases of Aspergillus oryzae Cs007

    Directory of Open Access Journals (Sweden)

    de Hong Yan

    2015-01-01

    Full Text Available In order to improve mycelium-bound ester hydrolases activities of Aspergillus oryzae Cs007, the main production conditions were investigated. The ester hydrolases activities were simultaneously determined by titration assay and spectrophotometric assay methods, using olive oil and p-nitrophenyl esters as substrates, respectively. The optimum carbon source and nitrogen source were olive oil and peptone, with the concentrations of 1% and 2.2%, respectively. The effects of carbon source, nitrogen source and their concentrations on the production of enzymes were identical when the enzymes activities were assayed by the two methods. The mycelium-bound enzymes showed hydrolytic activity toward all the tested p-nitrophenyl esters, triglycerides and fatty acid ethyl esters. But it showed greater preference for long-chain triglycerides and short-chain p-nitrophenyl esters.

  13. Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

    Science.gov (United States)

    Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids

    Directory of Open Access Journals (Sweden)

    Nilakshi Jayawardena

    2015-01-01

    Full Text Available The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P<0.05 increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P<0.05 reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities.

  15. [Soil hydrolase characteristics in late soil-thawing period in subalpine/alpine forests of west Sichuan].

    Science.gov (United States)

    Tan, Bo; Wu, Fu-Zhong; Yang, Wan-Qin; Yu, Sheng; Yang, Yu-Lian; Wang, Ao

    2011-05-01

    Late soil-thawing period is a critical stage connecting winter and growth season. The significant temperature fluctuation at this stage might have strong effects on soil ecological processes. In order to understand the soil biochemical processes at this stage in the subalpine/alpine forests of west Sichuan, soil samples were collected from the representative forests including primary fir forest, fir and birch mixed forest, and secondary fir forest in March 5-April 25, 2009, with the activities of soil invertase, urease, and phosphatase (neutral, acid and alkaline phosphatases) measured. In soil frozen period, the activities of the three enzymes in test forests still kept relatively higher. With the increase of soil temperature, the activities of hydrolases at the early stage of soil-thawing decreased rapidly after a sharp increase, except for neutral phosphatease. Thereafter, there was an increase in the activities of urease and phosphatase. Relative to soil mineral layer, soil organic layer had higher hydrolase activity in late soil-thawing period, and showed more obvious responses to the variation of soil temperature.

  16. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

    Directory of Open Access Journals (Sweden)

    Angsana Keeratijarut

    Full Text Available The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1. This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5. Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

  17. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

    Science.gov (United States)

    Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

    2008-02-01

    The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

  18. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats

    NARCIS (Netherlands)

    Koeners, Maarten P.; Wesseling, Sebastiaan; Ulu, Arzu; Lopez Sepulveda, Rocio; Morisseau, Christophe; Braam, Branko; Hammock, Bruce D.; Joles, Jaap A.

    Koeners MP, Wesseling S, Ulu A, Sepulveda RL, Morisseau C, Braam B, Hammock BD, Joles JA. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats. Am J Physiol Endocrinol Metab 300: E691-E698, 2011. First published January 25, 2011; doi:

  19. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  20. Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

    Science.gov (United States)

    Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

  1. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    Science.gov (United States)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  2. Murein Hydrolase Activity in the Surface Layer of Lactobacillus acidophilus ATCC 4356▿

    OpenAIRE

    Prado Acosta, Mariano; Palomino, María Mercedes; Allievi, Mariana C.; Rivas, Carmen Sanchez; Ruzal, Sandra M.

    2008-01-01

    We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verificati...

  3. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  4. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.

    2015-03-31

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

  5. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    Science.gov (United States)

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

  6. Discovery of α-L-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Katayama, Takane; Arakawa, Takatoshi

    2017-01-01

    Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence...

  7. MR imaging of intracellular and extracellular deoxyhemoglobin

    International Nuclear Information System (INIS)

    Janick, P.A.; Grossman, R.I.; Asakura, T.

    1989-01-01

    MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

  8. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou

    2010-11-01

    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  9. Serum concentration of ubiquitin c-terminal hydrolase-L1 in detecting severity of traumatic brain injury

    Science.gov (United States)

    Siahaan, A. M. P.; Japardi, I.; Hakim, A. A.

    2018-03-01

    One of the main problems with ahead injury is assessing the severity. While physical examination and imaging had limitations, neuronal damage markers, ubiquitin C-terminal hydrolase-L1 (UCH-L1), released in theblood may provide valuable information about diagnosis the traumatic brain injury (TBI).Analyzing the concentrations of serum ubiquitin C-terminal hydrolase-L1 (UCH-L1), there must have a neuronal injury biomarker, in theTBI patients serum and their association with clinical characteristics and outcome. There were 80 TBI subjects, and there are mild, moderate, and severe involved in this study of case- control. By using ELISA, we studied the profile of serum UCH-L1 levels for TBI patients. TheUCH-L1 serum level of moderate and severe head injury is higher than in mild head injury (pinjury patients. There is no particular correlation found between serum UCH-L1 level and outcome. Serum levels of UCH-L1 appear to have potential clinical utility in diagnosing TBI but do not correlate with outcome.

  10. An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities.

    Science.gov (United States)

    Lee, Yian Hoon; Choo, Candy; Watawana, Mindani I; Jayawardena, Nilakshi; Waisundara, Viduranga Y

    2015-11-01

    Eighteen edible plants were assessed for their antioxidant potential based on oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics, vitamin C content and various lipophilic antioxidants. The inhibitory activities of the plant extracts against the enzymatic activities of α-amylase and α-glucosidase were also evaluated. The antioxidant and starch hydrolase activities of the plants varied widely across a single batch of analysis. The ORAC and DPPH radical scavenging EC50 values varied between 298 and 1984 Trolox equivalents g(-1) fresh weight and between 91 and 533 mg kg(-1) fresh weight, respectively. The total phenolics and vitamin C contents varied between 32 and 125 mg gallic acid equivalents g(-1) fresh weight and between 96 and 285 µg g(-1) fresh weight, respectively. All the plants contained neoxanthin, violaxanthin, and α- and β-carotene in varying amounts. Coccinia grandis, Asparagus racemosus, Costus speciosus, Amaranthus viridis and Annona muricata displayed the highest inhibitory activities against starch hydrolases. They were the most efficient against the breakdown of seven starches exposed to the two enzymes as well. Overall, the edible plants were observed to display a high antioxidant potential with starch hydrolase inhibitory properties, which were beneficial in their being recognized as functional food. © 2014 Society of Chemical Industry.

  11. Single-cell intracellular nano-pH probes†

    OpenAIRE

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

  12. Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Manchi C.M.; Kuppan, Gokulan; Shetty, Nishant D.; Owen, Joshua L.; Ioerger, Thomas R.; Sacchettini, James C. (TAM)

    2009-12-01

    S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3{prime}-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3{prime} hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 {angstrom} resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5{prime} hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

  13. The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten

    2010-01-01

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto......Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria...... of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S...

  14. Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism

    DEFF Research Database (Denmark)

    Kory, Nora; Grond, Susanne; Kamat, Siddhesh S

    2017-01-01

    Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids...

  15. Intracellular Erythrocyte Platelet-activating Factor Acetylhydrolase I Inactivates Aspirin in Blood*

    Science.gov (United States)

    Zhou, Gang; Marathe, Gopal K.; Willard, Belinda; McIntyre, Thomas M.

    2011-01-01

    Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A2 with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A2 synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood. PMID:21844189

  16. Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood.

    Science.gov (United States)

    Zhou, Gang; Marathe, Gopal K; Willard, Belinda; McIntyre, Thomas M

    2011-10-07

    Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.

  17. Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

    Czech Academy of Sciences Publication Activity Database

    Čtrnáctá, Vlasta; Fritzler, J. M.; Šurínová, M.; Hrdý, I.; Zhu, G.; Stejskal, F.

    2010-01-01

    Roč. 126, č. 2 (2010), s. 113-116 ISSN 0014-4894 Institutional research plan: CEZ:AV0Z50520701 Keywords : S-adenosylhomocysteine hydrolase * D-eritadenine * (S)-DHPA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.869, year: 2010

  18. Genetic variation in the bleomycin hydrolase gene and bleomycin-induced pulmonary toxicity in germ cell cancer patients

    NARCIS (Netherlands)

    Nuver, J; Lutke-Holzik, MF; van Zweeden, M; Hoekstra, HJ; Meijer, C; Suurmeijer, AJH; Hofstra, RM; Sluiter, WJ; Sleijfer, D; Gietema, JA; Groen, Hendricus; Groen, Herman

    Objective Use of bleomycin as a cytotoxic agent is limited by its pulmonary toxicity. Bleomycin is mainly excreted by the kidneys, but can also be inactivated by bleomycin hydrolase (BMH). An 1450A > G polymorphic site in the BMH gene results in an amino acid substitution in the C-terminal domain of

  19. A Personal Retrospective: Elevating Anandamide (AEA by Targeting Fatty Acid Amide Hydrolase (FAAH and the Fatty Acid Binding Proteins (FABPs

    Directory of Open Access Journals (Sweden)

    Dale Deutsch

    2016-10-01

    Full Text Available This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and, until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and, as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that solubilize anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions.

  20. Potassium biphthalate buffer for pH control to optimize glycosyl hydrolase production in shake flasks using filamentous fungi

    Directory of Open Access Journals (Sweden)

    Patrícia dos Santos Costa

    Full Text Available Abstract The optimization of culture medium with statistical methods is widely used in filamentous fungi glycosyl hydrolase production. The implementation of such methodology in bioreactors is very expensive as it requires several pH-controlled systems operating in parallel in order to test a large number of culture media components. The objective of this study was to evaluate potassium biphthalate buffer for pH control, which allows the optimization studies to be performed in shake flasks.The results have shown that buffering the culture medium with 0.1 M potassium biphthalate allowed pH control, resulting in a decrease of the standard deviation of triplicates for pH and activities of glycosyl hydrolase measurements. The use of this buffer allowed shake flask culture media optimization of enzyme production by Trichoderma harzianum, increasing the cellulase activity by more than 2 times compared to standard unbuffered culture medium. The same buffer can be used for culture media optimization of other fungi, such as Penicillium echinulatum.

  1. Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice

    OpenAIRE

    Murthy, Vishakantha; Reyes, Santiago; Geng, Liyi; Gao, Yang; Brimijoin, Stephen

    2016-01-01

    Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains...

  2. Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

    Science.gov (United States)

    Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

    2018-06-04

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

  3. Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.

    Directory of Open Access Journals (Sweden)

    Xiaojing Jia

    Full Text Available Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A and GH67 α-glucuronidase (Agu67A from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

  4. Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion

    Directory of Open Access Journals (Sweden)

    Jay E. Mellon

    2015-08-01

    Full Text Available Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates.

  5. Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

    Science.gov (United States)

    Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

    2016-06-01

    Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Enhanced intracellular delivery and antibacterial efficacy of enrofloxacin-loaded docosanoic acid solid lipid nanoparticles against intracellular Salmonella.

    Science.gov (United States)

    Xie, Shuyu; Yang, Fei; Tao, Yanfei; Chen, Dongmei; Qu, Wei; Huang, Lingli; Liu, Zhenli; Pan, Yuanhu; Yuan, Zonghui

    2017-01-23

    Enrofloxacin-loaded docosanoic acid solid lipid nanoparticles (SLNs) with different physicochemical properties were developed to enhance activity against intracellular Salmonella. Their cellular uptake, intracellular elimination and antibacterial activity were studied in RAW 264.7 cells. During the experimental period, SLN-encapsulated enrofloxacin accumulated in the cells approximately 27.06-37.71 times more efficiently than free drugs at the same extracellular concentration. After incubation for 0.5 h, the intracellular enrofloxacin was enhanced from 0.336 to 1.147 μg/mg of protein as the sizes of nanoparticles were increased from 150 to 605 nm, and from 0.960 to 1.147 μg/mg of protein when the charge was improved from -8.1 to -24.9 mv. The cellular uptake was more significantly influenced by the size than it was by the charge, and was not affected by whether the charge was positive or negative. The elimination of optimal SLN-encapsulated enrofloxacin from the cells was significantly slower than that of free enrofloxacin after removing extracellular drug. The inhibition effect against intracellular Salmonella CVCC541 of 0.24 and 0.06 μg/mL encapsulated enrofloxacin was stronger than 0.6 μg/mL free drug after all of the incubation periods and at 48 h, respectively. Docosanoic acid SLNs are thus considered as a promising carrier for intracellular bacterial treatment.

  7. Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes

    Directory of Open Access Journals (Sweden)

    Pascal Viens

    2015-10-01

    Full Text Available Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC or chitosan oligosaccharides (CHOS from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

  8. N (6-substituted AMPs inhibit mammalian deoxynucleotide N-hydrolase DNPH1.

    Directory of Open Access Journals (Sweden)

    Claire Amiable

    Full Text Available The gene dnph1 (or rcl encodes a hydrolase that cleaves the 2'-deoxyribonucleoside 5'-monophosphate (dNMP N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N (6-substituted AMPs, and among them, cytotoxic cytokinin riboside 5'-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.

  9. Self-organization of intracellular gradients during mitosis

    Directory of Open Access Journals (Sweden)

    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  10. Evaluation of the Stability of the Total Antioxidant Capacity, Polyphenol Contents, and Starch Hydrolase Inhibitory Activities of Kombucha Teas Using an In Vitro Model of Digestion

    Directory of Open Access Journals (Sweden)

    Mindani I. Watawana

    2015-01-01

    Full Text Available The objective of this study was to evaluate and compare antioxidant and starch hydrolase inhibitory activity of three different types of Kombucha beverages prepared by three pellicles with different microbial compositions. The fermentation process was carried out for 7 days and the assessments of antioxidant and starch hydrolase inhibitory activities as well as tea phenolic compounds were carried out. These parameters were also evaluated after subjecting the final fermented samples to gastric and duodenal digestion in an in vitro digestion model. The pH had a statistically significant decrease during the period of fermentation. The total phenolics content and antioxidant activities had increased during the fermentation process as well as when subjected to digestion. The starch hydrolase inhibitory activities also increased in a similar manner during the different phases. The α-amylase and α-glucosidase inhibitory activities showed statistically significant increases (P<0.05 as the fermentation progressed, while an increase was observed after being subjected to pancreatic and duodenal digestion as well. All three types of tea showed a higher α-amylase inhibitory activity than α-glucosidase inhibitory activity.

  11. Some hydrolase activities from the tick Hyalomma lusitanicum Koch, 1844 (Ixodoidea: Ixodida

    Directory of Open Access Journals (Sweden)

    Giménez-Pardo C.

    2008-12-01

    Full Text Available In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

  12. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

    Science.gov (United States)

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

  13. The Vital Function of Fe3O4@Au nanocomposites for Hydrolase Biosensor Design and Its Application in Detection of Methyl Parathion

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yuting; Zhang, Weiying; Lin, Yuehe; Du, Dan

    2013-02-04

    A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned by OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng/mL and a detection limit of 0.1 ng/mL. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.

  14. Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

    NARCIS (Netherlands)

    Rozeboom, Henriëtte J.; Yu, Shukun; Madrid, Susan; Kalk, Kor H.; Zhang, Ran; Dijkstra, Bauke W.

    2013-01-01

    α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades

  15. Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tasuku; Saikawa, Kyo [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Kim, Seonah [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Fujita, Kiyotaka [Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima (Japan); Ishiwata, Akihiro [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); Kaeothip, Sophon [ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Arakawa, Takatoshi; Wakagi, Takayoshi [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Beckham, Gregg T., E-mail: Gregg.Beckham@nrel.gov [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Ito, Yukishige [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Fushinobu, Shinya, E-mail: asfushi@mail.ecc.u-tokyo.ac.jp [Department of Biotechnology, The University of Tokyo, Tokyo (Japan)

    2014-04-25

    Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

  16. Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

    Science.gov (United States)

    Pushparajah, Daphnee S; Umachandran, Meera; Plant, Kathryn E; Plant, Nick; Ioannides, Costas

    2007-02-28

    The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

  17. Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol

    Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til oligosakkarider, som potentielt kan fungere som prebiotika ved at stimulere væksten af...... omfatter karakterisering af de producerede enzymer samt cDNA kloning af formodet GH61 endo Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til...

  18. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Xianglu Li

    2009-01-01

    Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

  19. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    Science.gov (United States)

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  20. Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

    2008-07-08

    Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

  1. Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

    Science.gov (United States)

    Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

    2009-10-26

    The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

  2. Construction and characterisation of a genetically engineered Escherichia coli strain for the epoxide hydrolase-catalysed kinetic resolution of epoxides

    NARCIS (Netherlands)

    Visser, H.; Oliveira Vil Filho, de M.; Liese, A.; Weijers, C.A.G.M.; Verdoes, J.C.

    2003-01-01

    The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell

  3. Ubiquitin C-Terminal Hydrolase-Activity Is Involved in Sperm Acrosomal Function and Anti-polyspermy Defense During Porcine Fertilization

    Czech Academy of Sciences Publication Activity Database

    Yi, Y. J.; Manandhar, G.; Sutovsky, M.; Rongfeng, L.; Jonáková, Věra; Oko, R.; Park, C. S.; Prather, R.S.; Sutovsky, P.

    2007-01-01

    Roč. 77, č. 5 (2007), s. 780-793 ISSN 0006-3363 R&D Projects: GA ČR GA303/06/0895; GA MŠk 1M06011 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701 Keywords : Ubiquitin * proteasome * hydrolase * spermadhesin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.670, year: 2007

  4. Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

    CSIR Research Space (South Africa)

    Botes, AL

    2005-01-01

    Full Text Available Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heath land indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2, 2-disubstituted epoxides...

  5. Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

    Science.gov (United States)

    Brulc, Jennifer M; Antonopoulos, Dionysios A; Miller, Margret E Berg; Wilson, Melissa K; Yannarell, Anthony C; Dinsdale, Elizabeth A; Edwards, Robert E; Frank, Edward D; Emerson, Joanne B; Wacklin, Pirjo; Coutinho, Pedro M; Henrissat, Bernard; Nelson, Karen E; White, Bryan A

    2009-02-10

    The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

  6. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  7. A proton wire and water channel revealed in the crystal structure of isatin hydrolase

    DEFF Research Database (Denmark)

    Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan Kristian

    2014-01-01

    to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only...... when the product is formed. The functional proton wire present in IH-b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification...

  8. HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

    NARCIS (Netherlands)

    STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

    1993-01-01

    It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

  9. Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

    Science.gov (United States)

    Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

    2017-01-01

    Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

  10. 4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

    NARCIS (Netherlands)

    Gangoiti Muñecas, Joana; van Leeuwen, Sander S; Gerwig, Gerrit J; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes,

  11. Overexpression of fatty acid amide hydrolase induces early flowering in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Neal D. Teaster

    2012-02-01

    Full Text Available N-Acylethanolamines (NAEs are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE. In animal systems this reaction is part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH, which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440 lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA, enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD and inductive long day (LD conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3, were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

  12. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice

    OpenAIRE

    Walentiny, D. Matthew; Vann, Robert E.; Wiley, Jenny L.

    2015-01-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ9 -tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with sim...

  13. Characterization of Leptin Intracellular Trafficking

    Directory of Open Access Journals (Sweden)

    E Walum

    2009-12-01

    Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

  14. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  15. Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

    NARCIS (Netherlands)

    Visser, H.; Weijers, C.A.G.M.; Ooyen, van A.J.J.; Verdoes, J.C.

    2002-01-01

    Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The

  16. Stereoselectivity and substrate specificity in the kinetic resolution of methyl-substituted 1-oxaspiro[2.5]octanes by Rhodotorula glutinis epoxide hydrolase

    NARCIS (Netherlands)

    Weijers, C.A.G.M.; Meeuwse, P.; Herpers, R.L.J.M.; Franssen, M.C.R.; Sudhölter, E.J.R.

    2005-01-01

    [GRAPHICS] The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the

  17. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    Science.gov (United States)

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  18. Exploring anti-bacterial compounds against intracellular Legionella.

    Directory of Open Access Journals (Sweden)

    Christopher F Harrison

    Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

  19. Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

    Czech Academy of Sciences Publication Activity Database

    Archelas, A.; Zhao, W.; Faure, B.; Iacazio, G.; Kotík, Michael

    2016-01-01

    Roč. 591, FEB 2016 (2016), s. 66-75 ISSN 0003-9861 Institutional support: RVO:61388971 Keywords : Catalytic mechanism * Epoxide hydrolase * Electrophilic catalysis Subject RIV: CE - Biochemistry Impact factor: 3.165, year: 2016

  20. Intracellular calcium levels can regulate Importin-dependent nuclear import

    International Nuclear Information System (INIS)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-01-01

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery

  1. Intracellular calcium levels can regulate Importin-dependent nuclear import

    Energy Technology Data Exchange (ETDEWEB)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  2. Expression of Nudix hydrolase genes in barley under UV irradiation

    Science.gov (United States)

    Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

    Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

  3. Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels

    Science.gov (United States)

    Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie HD

    2015-01-01

    Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production. DOI: http://dx.doi.org/10.7554/eLife.05896.001 PMID:25647728

  4. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  5. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jennifer Hurst-Kennedy

    2012-01-01

    Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

  6. Single-cell intracellular nano-pH probes†

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  7. Single-cell intracellular nano-pH probes.

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  8. Mycobacterium intracellulare Infection Mimicking Progression of Scleroderma

    DEFF Research Database (Denmark)

    Krabbe, Simon; Engelhart, Merete; Thybo, Sören

    2017-01-01

    This case report describes a patient with scleroderma who developed Mycobacterium intracellulare infection, which for more than a year mimicked worsening of her connective tissue disorder. The patient was diagnosed with scleroderma based on puffy fingers that developed into sclerodactyly, abnormal......, unfortunately with significant scarring. Immunodeficiency testing was unremarkable. In summary, an infection with Mycobacterium intracellulare was mistaken for an unusually severe progression of scleroderma....

  9. Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

    Science.gov (United States)

    Higgins, Melanie A; Whitworth, Garrett E; El Warry, Nahida; Randriantsoa, Mialy; Samain, Eric; Burke, Robert D; Vocadlo, David J; Boraston, Alisdair B

    2009-09-18

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  10. Uptake and intracellular activity of AM-1155 in phagocytic cells.

    Science.gov (United States)

    Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

    1996-01-01

    The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

  11. First glycoside hydrolase family 2 enzymes from Thermus antranikianii and Thermus brockianus with β-glucosidase activity

    Directory of Open Access Journals (Sweden)

    Carola eSchröder

    2015-06-01

    Full Text Available Two genes tagh2 and tbgh2 coding for enzymes with hydrolytic activity towards esculin were identified from the extreme thermophilic, aerobic bacteria Thermus antranikianii (Ta and T. brockianus (Tb. Shortened conserved domains predicted a membership of the enzymes of glycoside hydrolase (GH family 2. At present, β-galactosidase activity is found frequently in GH family 2 but β-glucosidase activity has not been reported in this family before. The enzymes TaGH2 and TbGH2 preferred hydrolysis of nitrophenol-linked β-D-glucopyranosides with specific activities of 3,966 U/mg and 660 U/mg, respectively. Residual activities of 40 % (TaGH2 and 51 % (TbGH2 towards 4-NP-β-D-galactopyranoside were observed. Furthermore, TaGH2 hydrolyzed cellobiose. TbGH2, however, showed no activity on cellobiose or lactose. The enzymes exhibited highest activity at 95 °C (TaGH2 and 90 °C (TbGH2 at pH 6.5. Both enzymes were extremely thermostable and thermal activation up to 250 % was observed at temperatures between 50 and 60 °C. Accordingly, the first thermoactive glycoside hydrolase family 2 enzymes with β glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to the enzymes of GH family 2.

  12. COMPARATIVE MODELLING AND LIGAND BINDING SITE PREDICTION OF A FAMILY 43 GLYCOSIDE HYDROLASE FROM Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Shadab Ahmed

    2012-06-01

    Full Text Available The phylogenetic analysis of Clostridium thermocellum family 43 glycoside hydrolase (CtGH43 showed close evolutionary relation with carbohydrate binding family 6 proteins from C. cellulolyticum, C. papyrosolvens, C. cellulyticum, and A. cellulyticum. Comparative modeling of CtGH43 was performed based on crystal structures with PDB IDs 3C7F, 1YIF, 1YRZ, 2EXH and 1WL7. The structure having lowest MODELLER objective function was selected. The three-dimensional structure revealed typical 5-fold beta–propeller architecture. Energy minimization and validation of predicted model with VERIFY 3D indicated acceptability of the proposed atomic structure. The Ramachandran plot analysis by RAMPAGE confirmed that family 43 glycoside hydrolase (CtGH43 contains little or negligible segments of helices. It also showed that out of 301 residues, 267 (89.3% were in most favoured region, 23 (7.7% were in allowed region and 9 (3.0% were in outlier region. IUPred analysis of CtGH43 showed no disordered region. Active site analysis showed presence of two Asp and one Glu, assumed to form a catalytic triad. This study gives us information about three-dimensional structure and reaffirms the fact that it has the similar core 5-fold beta–propeller architecture and so probably has the same inverting mechanism of action with the formation of above mentioned catalytic triad for catalysis of polysaccharides.

  13. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

    International Nuclear Information System (INIS)

    Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

    2014-01-01

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

  14. Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

    DEFF Research Database (Denmark)

    Tagami, Takayoshi; Okuyama, Masayuki; Nakai, Hiroyuki

    2013-01-01

    Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers...

  15. The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Tisch Doris

    2011-12-01

    Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

  16. Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

    Science.gov (United States)

    Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

    2012-01-15

    Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

    Science.gov (United States)

    Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

    2017-10-23

    Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  18. Microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease: A comprehensive meta-analysis

    OpenAIRE

    LI, HUI; FU, WEI-PING; HONG, ZE-HUI

    2012-01-01

    Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the detoxification the products of smoking and is proposed to be a genetic factor for the development of chronic obstructive pulmonary disease (COPD). Two functional polymorphisms of EPHX1, T113C and A139G, have been analyzed in numerous studies to assess the COPD risk attributed to these variants. However, the conclusions were controversial. We performed a comprehensive meta-analysis to clarify these findings. A total of 24 studie...

  19. Influence of intracellular acidosis on contractile function in the working rat heart

    International Nuclear Information System (INIS)

    Jeffrey, F.M.H.; Malloy, C.R.; Radda, G.K.

    1987-01-01

    The decrease in myocardial contractility during ischemia, hypoxia, and extracellular acidosis has been attributed to intracellular acidosis. Previous studies of the relationship between pH and contractile state have utilized respiratory or metabolic acidosis to alter intracellular pH. The authors developed a model in the working perfused rat heart to study the effects of intracellular acidosis with normal external pH and optimal O 2 delivery. Intracellular pH and high-energy phosphates were monitored by 31 P nuclear magnetic resonance spectroscopy. Hearts were perfused to a steady state with a medium containing 10 mM NH 4 Cl. Acidosis induced a substantial decrease in aortic flow and stroke volume which was associated with little change in peak systolic pressure. It was concluded that (1) for the same intracellular acidosis the influence on tension development was more pronounced with a combined extra- and intracellular acidosis than with an isolated intracellular acidosis, and (2) stroke volume at constant preload was impaired by intracellular acidosis even though changes in developed pressure were minimal. These observations suggest that isolated intracellular acidosis has adverse effects on diastolic compliance and/or relaxation

  20. Intracellular trafficking of new anticancer therapeutics: antibody–drug conjugates

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody–drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs. PMID:28814834

  1. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

  2. Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

    Science.gov (United States)

    Hafner, Anne E.; Rieger, Heiko

    2018-03-01

    The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

  3. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina

    Directory of Open Access Journals (Sweden)

    Jang Hoon Kim

    2015-12-01

    Full Text Available Selaginellin derivatives 1–3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1–3 inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1–3 and sEH were solved by docking simulations. According to quantitative analysis, 1–3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  4. Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Fushinobu, Shinya; Hidaka, Masafumi; Wakagi, Takayoshi; Shoun, Hirofumi; Taniguchi, Hajime; Kitaoka, Motomitsu

    2008-04-01

    The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

  5. Molecular Cloning and Nucleotide Sequence of the Gene Encoding the Major Peptidoglycan Hydrolase of Lactococcus lactis, a Muramidase Needed for Cell Separation

    NARCIS (Netherlands)

    Buist, Girbe; Kok, Jan; Leenhouts, Kees J.; Dabrowska, Magdalena; Venema, Gerhardus; Haandrikman, Alfred J.

    A gene of Lactococcus lactis subsp, cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells,

  6. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

    Directory of Open Access Journals (Sweden)

    Farhana R. Pinu

    2017-10-01

    Full Text Available Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  7. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

    Directory of Open Access Journals (Sweden)

    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  8. A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

    Science.gov (United States)

    2000-03-31

    have a major impact on neuronal intracellular signaling. Most of the ER in neurons and glia appears to accumulate calcium by energy driven ion pumps...secretion of exocrine, endocrine, and neurocrine products, regulation of glycogenolysis and gluconeogenesis , intracellular transport, secretion of fluids...the RyRs [140]. Furthermore, the intracellular expression of these receptor-channels in neuronal ER is also reciprocal with RyRs located primarily in

  9. ETHANOL PRECIPITATION OF GLYCOSYL HYDROLASES PRODUCED BY Trichoderma harzianum P49P11

    Directory of Open Access Journals (Sweden)

    M. A. Mariño

    2015-06-01

    Full Text Available AbstractThis study aimed to concentrate glycosyl hydrolases produced by Trichoderma harzianum P49P11 by ethanol precipitation. The variables tested besides ethanol concentration were temperature and pH. The precipitation with 90% (v/v ethanol at pH 5.0 recovered more than 98% of the xylanase activity, regard less of the temperature (5.0, 15.0, or 25.0 °C. The maximum recovery of cellulase activity as FPase was 77% by precipitation carried out at this same pH and ethanol concentration but at 5.0 °C. Therefore, ethanol precipitation can be considered to be an efficient technique for xylanase concentration and, to a certain extent, also for the cellulase complex.

  10. The use of neutron scattering to determine the functional structure of glycoside hydrolase.

    Science.gov (United States)

    Nakamura, Akihiko; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko

    2016-10-01

    Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Hydrolytic enzymes in the central vacuole of plant cells

    International Nuclear Information System (INIS)

    Boller, T.; Kende, H.

    1979-01-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β-N-acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter

  12. Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M; Skovbjerg, H; Norén, Ove

    1984-01-01

    of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence...

  13. Nuclear magnetic resonance studies of intracellular ions in perfused from heart

    International Nuclear Information System (INIS)

    Burnstein, D.; Fossel, E.T.

    1987-01-01

    Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 μM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in changes in the intracellular sodium levels. An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR method. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate

  14. Intracellular pH homeostasis in Leishmania donovani amastigotes and promastigotes

    International Nuclear Information System (INIS)

    Glaser, T.A.; Baatz, J.E.; Kreishman, G.P.; Mukkada, A.J.

    1988-01-01

    Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31 P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment

  15. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    Science.gov (United States)

    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  16. Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

    Science.gov (United States)

    Haroune, Lounès; Saibi, Sabrina; Cabana, Hubert; Bellenger, Jean-Philippe

    2017-01-17

    The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

  17. Hepatic cholesterol ester hydrolase in human liver disease.

    Science.gov (United States)

    Simon, J B; Poon, R W

    1978-09-01

    Human liver contains an acid cholesterol ester hydrolase (CEH) of presumed lysosomal origin, but its significance is unknown. We developed a modified CEH radioassay suitable for needle biopsy specimens and measured hepatic activity of this enzyme in 69 patients undergoing percutaneous liver biopsy. Histologically normal livers hydrolyzed 5.80 +/- 0.78 SEM mumoles of cholesterol ester per hr per g of liver protein (n, 10). Values were similar in alcoholic liver disease (n, 17), obstructive jaundice (n, 9), and miscellaneous hepatic disorders (n, 21). In contrast, mean hepatic CEH activity was more than 3-fold elevated in 12 patients with acute hepatitis, 21.05 +/- 2.45 SEM mumoles per hr per g of protein (P less than 0.01). In 2 patients studied serially, CEH returned to normal as hepatitis resolved. CEH activity in all patients paralleled SGOT levels (r, 0.84; P less than 0.01). There was no correlation with serum levels of free or esterified cholesterol nor with serum activity of lecithin-cholesterol acyltransferase, the enzyme responsible for cholesterol esterification in plasma. These studies confirm the presence of CEH activity in human liver and show markedly increased activity in acute hepatitis. The pathogenesis and clinical significance of altered hepatic CEH activity in liver disease require further study.

  18. GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

    Directory of Open Access Journals (Sweden)

    Naumoff Daniil G

    2005-08-01

    Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

  19. Ortholog-based screening and identification of genes related to intracellular survival.

    Science.gov (United States)

    Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

    2018-04-20

    Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

  20. DMPD: Intracellular DNA sensors in immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18573338 Intracellular DNA sensors in immunity. Takeshita F, Ishii KJ. Curr Opin Im...munol. 2008 Aug;20(4):383-8. Epub 2008 Jun 23. (.png) (.svg) (.html) (.csml) Show Intracellular DNA sensors ...in immunity. PubmedID 18573338 Title Intracellular DNA sensors in immunity. Authors Takeshita F, Ishii KJ. P

  1. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually

  2. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    Science.gov (United States)

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  3. The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

    OpenAIRE

    Kreuzer, Johannes

    2015-01-01

    The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

  4. Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

    Science.gov (United States)

    Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P.; Kemper, Claudia

    2013-01-01

    Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  5. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

    Science.gov (United States)

    Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

    2018-03-20

    Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

  6. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

  7. The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

    Science.gov (United States)

    Johannes, Ludger

    2002-07-01

    Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

  8. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    Science.gov (United States)

    2015-06-19

    decontamination strategies>> Maryline DEFEZ 1𔃼, Melissa HUNTER3J Susan WELKOS :~J Christopher COTE3 1 University Grenoble-Alpes, Grenoble, France. 1...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a...8217 • Accidentally in Humans • Natural reservoir is soil • Anthrax Disease Cycle: - animals infected by soilborne spores in food and water or bites from certain

  9. New perspective in the assessment of total intracellular magnesium

    Directory of Open Access Journals (Sweden)

    Azzurra Sargenti

    2014-01-01

    Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

  10. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai; Nicora, Carrie D.; Aldrich, Joshua T.; Frey, Dario; Kolinko, Sebastian; Robinson, Errol W.; Jacobs, Jon M.; Adams, Paul D.; Northen, Trent R.; Simmons, Blake A.; Singer, Steven W.

    2016-08-23

    ABSTRACT

    Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.

    IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose

  11. Advances in genetic manipulation of obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  12. Intracellular transport of fat-soluble vitamins A and E.

    Science.gov (United States)

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

  13. Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

    International Nuclear Information System (INIS)

    Akbari, Vajihe; Abedi, Daryoush; Pardakhty, Abbas; Sadeghi-Aliabadi, Hojjat

    2013-01-01

    In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300–600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

  14. Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Akbari, Vajihe; Abedi, Daryoush [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of); Pardakhty, Abbas [Kerman University of Medical Sciences, Pharmaceutics Research Center (Iran, Islamic Republic of); Sadeghi-Aliabadi, Hojjat, E-mail: sadeghi@pharm.mui.ac.ir [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of)

    2013-04-15

    In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300-600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

  15. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    Science.gov (United States)

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird?s-Nest Fern

    OpenAIRE

    Wu, Guan-Long; Kuo, Tzu-Hao; Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nem...

  17. Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database : The Lactobacillus reuteri NCC 2613 GtfB

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S.; Meng, Xiangfeng; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving

  18. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    Science.gov (United States)

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  19. Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum.

    Science.gov (United States)

    Cibik, R; Tailliez, P; Langella, P; Chapot-Chartier, M P

    2001-02-01

    A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.

  20. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Gregg, Katie J.; Boraston, Alisdair B.

    2009-01-01

    The catalytic module of a family 101 glycoside hydrolase from S. pneumoniae was cloned, recombinantly produced and crystallized. Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-d-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2 1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 Å, β = 112.46°. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl) -α-d-galactopyranoside, which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase

  1. A bacteriophage endolysin that eliminates intracellular streptococci

    Science.gov (United States)

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  2. Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two beta-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol; Nakai, Hiroyuki; Gotfredsen, Charlotte Held

    2011-01-01

    Two beta-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k...

  3. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

    Science.gov (United States)

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

  4. 1,3-Disubstituted Ureas Functionalized with Ether Groups are Potent Inhibitors of the Soluble Epoxide Hydrolase with Improved Pharmacokinetic Properties

    OpenAIRE

    Kim, In-Hae; Tsai, Hsing-Ju; Nishi, Kosuke; Kasagami, Takeo; Morisseau, Christophe; Hammock, Bruce D.

    2007-01-01

    Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokineti...

  5. The interferon response to intracellular DNA: why so many receptors?

    Science.gov (United States)

    Unterholzner, Leonie

    2013-11-01

    The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. Copyright © 2013 Elsevier GmbH. All rights reserved.

  6. Epoxide hydrolase affects estrogen production in the human ovary.

    Science.gov (United States)

    Hattori, N; Fujiwara, H; Maeda, M; Fujii, S; Ueda, M

    2000-09-01

    To investigate the mechanisms of ovarian cell differentiation, we raised a new monoclonal antibody, HCL-3, which reacted with human luteal cells. It also reacted with human and porcine hepatocytes. The immunoaffinity-purified HCL-3 antigen from human corpora lutea (CL) was shown to be a 46-kDa protein. The N-terminal 22 amino acids of the 46-kDa protein from porcine liver exhibited high homology (82%) to human microsomal epoxide hydrolase (mEH). The purified HCL-3 antigen from human CL or porcine liver showed EH enzyme activity, confirming that HCL-3 antigen is identical to mEH, which is reported to detoxify the toxic substrates in the liver. In human follicles, mEH was immunohistochemically detected on granulosa and theca interna cells. In the menstrual and pregnant CL, mEH was also expressed on large and small luteal cells. A competitive inhibitor of EH, 1,2-epoxy-3,3,3-trichloropropane, inhibited the conversion of estradiol from testosterone by granulosa cells cultured in vitro, indicating the involvement of mEH in ovarian estrogen production. Because anticonvulsant sodium valproate and its analogues were reported to inhibit EH enzyme activity, these findings provide a new insight into the etiology of endocrine disorders that are frequently observed among epileptic patients taking anticonvulsant drugs.

  7. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

    Science.gov (United States)

    Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

    2015-07-15

    Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

  8. The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

    Science.gov (United States)

    Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; Brink, Peter R.; White, Thomas W.; Mathias, Richard T.

    2013-01-01

    Purpose. Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. Methods. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer–based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na+ concentrations were measured by injecting the Na+-sensitive dye sodium-binding benzofuran isophthalate. Results. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na+ concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from −45 mV in central fiber cells to −60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Conclusions. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na+-leak channels in fiber cells of larger lenses. PMID:23211824

  9. Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

    Science.gov (United States)

    Ahmedov, Merdin Lyutviev; Kemerdere, Rahsan; Baran, Oguz; Inal, Berrin Bercik; Gumus, Alper; Coskun, Cihan; Yeni, Seher Naz; Eren, Bulent; Uzan, Mustafa; Tanriverdi, Taner

    2017-10-01

    We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r 2  = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r 2  = 0.7, P = 0.00001 and r 2  = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r 2  = 0.06, P = 0.00001). Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Ethanol production with a flocculating mutant of Zymomonas mobilis and immobilized glycoside hydrolases. Ethanolgewinnung mit einer flockenden Mutante von Zymomonas mobilis und immobilisierten Glycosidhydrolasen

    Energy Technology Data Exchange (ETDEWEB)

    Tramm-Werner, S.

    1987-05-25

    A method to extend the substrate spectrum of Z. mobilis was developed. Higher ethanol yields were achieved by simultaneous use of hydrolases cross-linked with glutar aldehyde together with the flocculating Zymomonas cells (TW 602). Apart from the high product yields, the method is characterized by low susceptibility to infections.

  11. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    Science.gov (United States)

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-05

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  12. Simultaneous Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Shares Discriminative Stimulus Effects with Δ9-Tetrahydrocannabinol in Mice

    OpenAIRE

    Hruba, Lenka; Seillier, Alexandre; Zaki, Armia; Cravatt, Benjamin F.; Lichtman, Aron H.; Giuffrida, Andrea; McMahon, Lance R.

    2015-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Δ9-tetrahydrocannabinol (Δ9-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N...

  13. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  14. Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

    Science.gov (United States)

    Okawa, Y; Yamaguchi, T

    1977-05-01

    1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.

  15. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  16. The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60 Å resolution.

    Science.gov (United States)

    Qi, Qi; Yang, Wen Jing; Zhou, Hu Jian; Ming, Deng Ming; Sun, Kai Lei; Xu, Tian Yu; Hu, Xiao Jian; Lv, Hong

    2017-07-01

    Zearalenone hydrolase (ZHD) is an α/β-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme-substrate complex structures have been reported in the resolution range 2.4-2.6 Å. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 Å resolution structure of a ZHD-product complex is reported which was determined from a C-terminally His 6 -tagged ZHD crystal soaked with 2 mM ZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD-substrate and ZHD-product structures show that the hydrophilic interactions change, especially Trp183 N ℇ1 , which shifts from contacting O2 to O12', suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

  17. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    Directory of Open Access Journals (Sweden)

    Beijing K. Huang

    2014-01-01

    Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

  18. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

    Science.gov (United States)

    Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

    2014-11-01

    TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  19. Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

    DEFF Research Database (Denmark)

    Ernstsen, Christina Lundgaard; Login, Frédéric H.; Jensen, Helene Halkjær

    2017-01-01

    Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacteria...

  20. Cationic Antimicrobial Peptide LL-37 Is Effective against both Extra- and Intracellular Staphylococcus aureus

    Science.gov (United States)

    Noore, Jabeen; Noore, Adly

    2013-01-01

    The increasing resistance of bacteria to conventional antibiotics and the challenges posed by intracellular bacteria, which may be responsible for chronic and recurrent infections, have driven the need for advanced antimicrobial drugs for effective elimination of both extra- and intracellular pathogens. The purpose of this study was to determine the killing efficacy of cationic antimicrobial peptide LL-37 compared to conventional antibiotics against extra- and intracellular Staphylococcus aureus. Bacterial killing assays and an infection model of osteoblasts and S. aureus were studied to determine the bacterial killing efficacy of LL-37 and conventional antibiotics against extra- and intracellular S. aureus. We found that LL-37 was effective in killing extracellular S. aureus at nanomolar concentrations, while lactoferricin B was effective at micromolar concentrations and doxycycline and cefazolin at millimolar concentrations. LL-37 was surprisingly more effective in killing the clinical strain than in killing an ATCC strain of S. aureus. Moreover, LL-37 was superior to conventional antibiotics in eliminating intracellular S. aureus. The kinetic studies further revealed that LL-37 was fast in eliminating both extra- and intracellular S. aureus. Therefore, LL-37 was shown to be very potent and prompt in eliminating both extra- and intracellular S. aureus and was more effective in killing extra- and intracellular S. aureus than commonly used conventional antibiotics. LL-37 could potentially be used to treat chronic and recurrent infections due to its effectiveness in eliminating not only extracellular but also intracellular pathogens. PMID:23274662

  1. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

    Science.gov (United States)

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-08-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

  2. The dead, hardened floral bracts of dispersal units of wild wheat function as storage for active hydrolases and in enhancing seedling vigor.

    Directory of Open Access Journals (Sweden)

    Buzi Raviv

    Full Text Available It is commonly assumed that the dead, hardened floral bracts of the dispersal unit of grasses have been evolved to protect seeds from predation and / or assist in fruit/caryopsis dispersal. While these structures have important agronomical and economical implications, their adaptive value has not been fully explored. We investigated the hypothesis that the maternally derived hardened floral bracts have been evolved not just as a means for caryopsis protection and dispersal, but also as storage for substances that might affect seed germination and seedling vigor. Dead glumes as well as lemmas and paleas of wild emmer wheat (Triticum turgidum var dicoccoides were found to store and release upon hydration active hydrolases including nucleases and chitinases. High nuclease activity was released upon hydration from glumes derived from wild strains of wheat including Triticum urartu and wild emmer wheat, while very low nuclease activity was detected in glumes derived from domesticated, free-threshing wheat cultivars (e.g., durum wheat. Germination from the intact dispersal unit of wild emmer wheat was delayed, but post germination growth was better than those of separated caryopses. Most notable was a significant increase in lateral root production on seedlings germinated from the intact dispersal unit. Proteome analysis of wild emmer wheat glumes revealed many proteins stored and released upon hydration including S1-type nucleases, peptidases, antifungal hydrolases such as chitinases and β-1,3-glucanase as well as pectin acetylesterase, a protein involved in cell wall degradation and remodeling. Also, reactive oxygen species (ROS-detoxifying enzymes such as superoxide dismutase and ascorbate peroxidase were overrepresented in dead glumes of wild emmer wheat. Thus our study highlighted previously unknown features of the dispersal unit in wild wheat in which the dead, hardened floral bracts enclosing the caryopsis store active hydrolases and

  3. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  4. Subversion of the cytoskeleton by intracellular bacteria: lessons from Listeria, Salmonella, and Vibrio

    Science.gov (United States)

    de Souza Santos, Marcela; Orth, Kim

    2018-01-01

    Summary Entry into host cells and intracellular persistence by invasive bacteria are tightly coupled to the ability of the bacterium to disrupt the eukaryotic cytoskeletal machinery. Herein we review the main strategies used by three intracellular pathogens to harness key modulators of the cytoskeleton. Two of these bacteria, namely Listeria monocytogenes and Salmonella enterica serovar Typhimurium, exhibit quite distinct intracellular lifestyles, and therefore, provide a comprehensive panel for the understanding of the intricate bacteria-cytoskeleton interplay during infections. The emerging intracellular pathogen Vibrio parahaemolyticus is depicted as a developing model for the uncovering of novel mechanisms used to hijack the cytoskeleton. PMID:25440316

  5. Tethering factors as organizers of intracellular vesicular traffic.

    Science.gov (United States)

    Yu, I-Mei; Hughson, Frederick M

    2010-01-01

    Intracellular trafficking entails the budding, transport, tethering, and fusion of transport vesicles and other membrane carriers. Here we review recent progress toward a mechanistic understanding of vesicle tethering. The known tethering factors are large complexes important for one or more intracellular trafficking pathways and are capable of interacting directly with many of the other principal components of the cellular trafficking machinery. Our review emphasizes recent developments in the in vitro reconstitution of vesicle tethering and the structural characterization of multisubunit tethering factors. The combination of these and other approaches has led to exciting progress toward understanding how these essential nanomachines work.

  6. Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

    Science.gov (United States)

    Guedes, A; Galuppo, L; Hood, D; Hwang, S H; Morisseau, C; Hammock, B D

    2017-05-01

    The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. In vitro experiments and clinical case series. sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. Absence of control group and evaluator blinding in case series. sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses. © 2016 EVJ Ltd.

  7. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Ji Hye; Joo, Sang-Woo [Department of Chemistry, Soongsil University, Seoul 156-743 (Korea, Republic of); Cho, Keunchang [Logos Biosystems, Incorporated, Anyang 431-070 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr, E-mail: sjoo@ssu.ac.kr [Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2011-06-10

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  8. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    International Nuclear Information System (INIS)

    Seo, Ji Hye; Joo, Sang-Woo; Cho, Keunchang; Lee, So Yeong

    2011-01-01

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  9. Fluorescent probes and nanoparticles for intracellular sensing of pH values

    Science.gov (United States)

    Shi, Wen; Li, Xiaohua; Ma, Huimin

    2014-12-01

    Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 - 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail.

  10. Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion.

    Science.gov (United States)

    Bharadwaj, Rajiv; Chen, Zhiwei; Datta, Supratim; Holmes, Bradley M; Sapra, Rajat; Simmons, Blake A; Adams, Paul D; Singh, Anup K

    2010-11-15

    The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidic-chip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose oligomers in around 1 min (10-fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotoga maritima . The results demonstrate that the cellulase is active at 80 °C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing.

  11. Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7

    Directory of Open Access Journals (Sweden)

    Jambunathan Niranjani

    2010-08-01

    Full Text Available Abstract Background Nudix hydrolases play a key role in maintaining cellular homeostasis by hydrolyzing various nuceloside diphosphate derivatives and capped mRNAs. Several independent studies have demonstrated that Arabidopsis nudix hydrolase 7 (AtNUDT7 hydrolyzes NADH and ADP-ribose. Loss of function Atnudt7-1 mutant plants (SALK_046441 exhibit stunted growth, higher levels of reactive oxygen species, enhanced resistance to pathogens. However, using the same T-DNA line, two other groups reported that mutant plants do not exhibit any visible phenotypes. In this study we analyze plausible factors that account for differences in the observed phenotypes in Atnudt7. Secondly, we evaluate the biochemical and molecular consequences of increased NADH levels due to loss of function of AtNUDT7 in Arabidopsis. Results We identified a novel conditional phenotype of Atnudt7-1 knockout plants that was contingent upon nutrient composition of potting mix. In nutrient-rich Metro-Mix, there were no phenotypic differences between mutant and wild-type (WT plants. In the nutrient-poor mix (12 parts vermiculite: 3 parts Redi-earth and 1 part sand, mutant plants showed the characteristic stunted phenotype. Compared with WT plants, levels of glutathione, NAD+, NADH, and in turn NADH:NAD+ ratio were higher in Atnudt7-1 plants growing in 12:3:1 potting mix. Infiltrating NADH and ADP-ribose into WT leaves was sufficient to induce AtNUDT7 protein. Constitutive over-expression of AtNudt7 did not alter NADH levels or resistance to pathogens. Transcriptome analysis identified nearly 700 genes differentially expressed in the Atnudt7-1 mutant compared to WT plants grown in 12:3:1 potting mix. In the Atnudt7-1 mutant, genes associated with defense response, proteolytic activities, and systemic acquired resistance were upregulated, while gene ontologies for transcription and phytohormone signaling were downregulated. Conclusions Based on these observations, we conclude that the

  12. Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

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    Jinhua Li

    2016-02-01

    Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

  13. Use of nanostructure initiator mass spectrometry (NIMS to deduce selectivity of reaction in glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Kai eDeng

    2015-10-01

    Full Text Available Chemically synthesized nanostructure-initiator mass spectrometry (NIMS probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

  14. Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries

    DEFF Research Database (Denmark)

    Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N.

    2015-01-01

    thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH......,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from....... The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies....

  15. Hydrolytic enzymes in the central vacuole of plant cells.

    Science.gov (United States)

    Boller, T; Kende, H

    1979-06-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha-mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar

  16. Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

    Science.gov (United States)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

    2017-08-01

    To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple

  17. Intracellular sodium hydrogen exchange inhibition and clinical myocardial protection.

    Science.gov (United States)

    Mentzer, Robert M; Lasley, Robert D; Jessel, Andreas; Karmazyn, Morris

    2003-02-01

    Although the mechanisms underlying ischemia/reperfusion injury remain elusive, evidence supports the etiologic role of intracellular calcium overload and oxidative stress induced by reactive oxygen species. Activation of the sodium hydrogen exchanger (NHE) is associated with intracellular calcium accumulation. Inhibition of the NHE-1 isoform may attenuate the consequences of this injury. Although there is strong preclinical and early clinical evidence that NHE inhibitors may be cardioprotective, definitive proof of this concept in humans awaits the results of ongoing clinical trials.

  18. The effect of sodium bicarbonate on intracellular pH using 31P-MR spectroscopy

    International Nuclear Information System (INIS)

    Nakashima, Kazuya; Kashiwagi, Shiro; Ito, Haruhide; Yamashita, Tetsuo; Kitahara, Tetsuhiro; Nakayama, Naoto; Saito, Kennichi

    1997-01-01

    This report deals with the effects of sodium bicarbonate on the intracellular pH of the brain and cerebral blood flow (CBF); five normal volunteers were studied. Intracellular pH and CBF were measured by phosphorus 31 magnetic resonance spectroscopy ( 31 P-MRS) and stable xenon computed tomography (Xe-CT), respectively. Each individual received 7% sodium bicarbonate (3.5 ml/kg body weight), infused intravenously over a 15-min period. Intracellular pH, CBF, and physiological parameters were determined before and after the injection. Intracellular pH was significantly decreased and CBF was increased. Among the physiological parameters, the hematocrit was significantly decreased and arterial pressure of carbon dioxide (PaCO 2 ), increased. These results suggest that increasing CO 2 contributes to the decrease in intracellular pH. In conclusion, three factors increase CBF during the administration of sodium bicarbonate to humans: arterial dilatation in response to carbon dioxide; decrease of the hematocrit, and intracellular cerebral acidosis. (author)

  19. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  20. Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

    Directory of Open Access Journals (Sweden)

    Walmor eDe Mello

    2015-01-01

    Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

  1. Fatty Acid Signaling: The New Function of Intracellular Lipases

    Directory of Open Access Journals (Sweden)

    Zuzana Papackova

    2015-02-01

    Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

  2. Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

    Science.gov (United States)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

    2017-10-01

    Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

  3. Fluorescent probes and nanoparticles for intracellular sensing of pH values

    International Nuclear Information System (INIS)

    Shi, Wen; Li, Xiaohua; Ma, Huimin

    2014-01-01

    Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 − 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail. (topical review)

  4. A method for functional trans-complementation of intracellular Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Shaun Steele

    Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

  5. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30-80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35-50 oC and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  6. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30–80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35–50 °C and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  7. Leishmania hijacking of the macrophage intracellular compartments.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Loiseau, Philippe M

    2016-02-01

    Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

  8. Monitorando atividades enzimáticas com sondas fluorogênicas

    Directory of Open Access Journals (Sweden)

    Caroline da Costa Silva Gonçalves

    2014-07-01

    Full Text Available Latent fluorogenic probes are essential tools for molecular and chemical biology, providing valuable information about enzymatic activity and occurrence. In this review, a brief outline of fluorophores and latent fluorogenic probes is given. Furthermore, advances and challenges in the development of fluorogenic chemical probes to visualize enzymatic activities (hydrolases and oxidoreductases of biotechnological and biomedical interest are highlighted, including some methodologies for intracellular imaging.

  9. pH-sensitive intracellular photoluminescence of carbon nanotube-fluorescein conjugates in human ovarian cancer cells

    International Nuclear Information System (INIS)

    Chen, M T; Ishikawa, F N; Gundersen, M A; Gomez, L M; Vernier, P T; Zhou, C

    2009-01-01

    To add to the understanding of the properties of functionalized carbon nanotubes in biological applications, we report a monotonic pH sensitivity of the intracellular fluorescence emission of single-walled carbon nanotube-fluorescein carbazide (SWCNT-FC) conjugates in human ovarian cancer cells. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. SWCNT-FC conjugates may serve as intracellular pH sensors.

  10. Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings.

    Science.gov (United States)

    Huang, A H; Moreau, R A; Liu, K D

    1978-03-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax).

  11. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

    Science.gov (United States)

    Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

    2017-01-01

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

  12. Structure-Based Optimization of Arylamides as Inhibitors of Soluble Epoxide Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Eldrup, Anne B.; Soleymanzadeh, Fariba; Taylor, Steven J.; Muegge, Ingo; Farrow, Neil A.; Joseph, David; McKellop, Keith; Man, Chuk C.; Kukulka, Alison; De Lombaert, Stephane; (Boehringer)

    2009-11-04

    Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations, the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotinamide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma exposure in the rat after oral administration.

  13. Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

    Science.gov (United States)

    Wang, Pan-Fen; Yep, Alejandra; Kenyon, George L; McLeish, Michael J

    2009-02-01

    Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.

  14. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-12-09

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

  15. DMPD: NOD-like receptors (NLRs): bona fide intracellular microbial sensors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18585455 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Shaw...tml) (.csml) Show NOD-like receptors (NLRs): bona fide intracellular microbial sensors. PubmedID 18585455 Ti...tle NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Authors

  16. A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

    Science.gov (United States)

    Maccarrone, M; Bari, M; Agrò, A F

    1999-02-15

    A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

  17. Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Fabian Arenas

    2017-11-01

    Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

  18. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  19. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-05-01

    The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

  20. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-01-01

    The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

  1. HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

    Science.gov (United States)

    Mehta, Kosha J; Farnaud, Sebastien; Patel, Vinood B

    2017-10-01

    In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p HFE and HAMP expressions were elevated only at low 1 g/L treatment (p HFE (p HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

  2. Evaluation of fish models of soluble epoxide hydrolase inhibition.

    Science.gov (United States)

    Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

    2001-01-01

    Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects. PMID:11171526

  3. Pathogenic mechanisms of intracellular bacteria.

    Science.gov (United States)

    Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

    2017-06-01

    We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

  4. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Khaled Alkhuder

    2009-01-01

    Full Text Available Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT. This gene (FTL_0766 was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

  5. Delivery of rifampicin-chitin nanoparticles into the intracellular compartment of polymorphonuclear leukocytes.

    Science.gov (United States)

    Smitha, K T; Nisha, N; Maya, S; Biswas, Raja; Jayakumar, R

    2015-03-01

    Polymorphonuclear leukocytes (PMNs) provide the primary host defence against invading pathogens by producing reactive oxygen species (ROS) and microbicidal products. However, few pathogens can survive for a prolonged period of time within the PMNs. Additionally their intracellular lifestyle within the PMNs protect themselves from the additional lethal action of host immune systems such as antibodies and complements. Antibiotic delivery into the intracellular compartments of PMNs is a major challenge in the field of infectious diseases. In order to deliver antibiotics within the PMNs and for the better treatment of intracellular bacterial infections we synthesized rifampicin (RIF) loaded amorphous chitin nanoparticles (RIF-ACNPs) of 350±50 nm in diameter. RIF-ACNPs nanoparticles are found to be non-hemolytic and non-toxic against a variety of host cells. The release of rifampicin from the prepared nanoparticles was ∼60% in 24 h, followed by a sustained pattern till 72 h. The RIF-ACNPs nanoparticles showed 5-6 fold enhanced delivery of RIF into the intracellular compartments of PMNs. The RIF-ACNPs showed anti-microbial activity against Escherichia coli, Staphylococcus aureus and a variety of other bacteria. In summary, our results suggest that RIF-ACNPs could be used to treat a variety of intracellular bacterial infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

    Directory of Open Access Journals (Sweden)

    Audrey Bernut

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

  7. Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in Lactobacillus casei.

    Science.gov (United States)

    Wang, Guohong; Yin, Sheng; An, Haoran; Chen, Shangwu; Hao, Yanling

    2011-08-01

    Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H(2)O(2)) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H(2)O(2)/min/10(8) colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H(2)O(2), survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10(5) CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.

  8. Distribution and function of carbamate hydrolase genes cehA and mcd in soils: the distinct role of soil pH.

    Science.gov (United States)

    Rousidou, Constantina; Karaiskos, Dionysis; Myti, Despoina; Karanasios, Evangelos; Karas, Panagiotis A; Tourna, Maria; Tzortzakakis, Emmanuel A; Karpouzas, Dimitrios G

    2017-01-01

    Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Model Linking Plasma and Intracellular Tenofovir/Emtricitabine with Deoxynucleoside Triphosphates.

    Directory of Open Access Journals (Sweden)

    Xinhui Chen

    Full Text Available The coformulation of the nucleos(tide analogs (NA tenofovir (TFV disoproxil fumarate (TDF and emtricitabine (FTC is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP and FTC-triphosphate (FTC-TP. Such complexities manifest in nonlinear intracellular pharmacokinetics (PK. In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD study among forty subjects receiving daily TDF/FTC (300 mg/200 mg from the first-dose to pharmacological intracellular steady-state (30 days. TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM. Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP and deoxycytidine triphosphate (dCTP production were 1020 fmol/106 cells (130% and 44.4 pmol/106 cells (82.5%, resulting in (90% prediction interval 11% (0.45%, 53% and 14% (2.6%, 35% reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP. Simulation

  10. Molecular detection and characterization of sustainable intracellular ...

    African Journals Online (AJOL)

    3Centre for Biopolymer and Bio-Molecular Research, Athlone College of Technology, Republic of Ireland. ... cells was associated with the elongation of micro-villar extension that ... Keywords: Intracellular contaminants, cell cultures, bacteria culture, pre-clinical studies. ... production work involving culture technology.

  11. Cationic polymers for intracellular delivery of proteins

    NARCIS (Netherlands)

    Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Samal, Sangram; Dubruel, Peter

    2015-01-01

    Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering

  12. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    thods of reduction of metal ions using plants or microorganisms are often ... have several advantages over bacteria, they are often pre- ferred. ... in static condition for a period of 7 days. ... work was focused on the production of intracellular gold.

  13. The effect of sodium bicarbonate on intracellular pH using {sup 31}P-MR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Kazuya; Kashiwagi, Shiro; Ito, Haruhide [Yamaguchi Univ., Ube (Japan). School of Medicine; Yamashita, Tetsuo; Kitahara, Tetsuhiro; Nakayama, Naoto; Saito, Kennichi

    1997-03-01

    This report deals with the effects of sodium bicarbonate on the intracellular pH of the brain and cerebral blood flow (CBF); five normal volunteers were studied. Intracellular pH and CBF were measured by phosphorus 31 magnetic resonance spectroscopy ({sup 31}P-MRS) and stable xenon computed tomography (Xe-CT), respectively. Each individual received 7% sodium bicarbonate (3.5 ml/kg body weight), infused intravenously over a 15-min period. Intracellular pH, CBF, and physiological parameters were determined before and after the injection. Intracellular pH was significantly decreased and CBF was increased. Among the physiological parameters, the hematocrit was significantly decreased and arterial pressure of carbon dioxide (PaCO{sub 2}), increased. These results suggest that increasing CO{sub 2} contributes to the decrease in intracellular pH. In conclusion, three factors increase CBF during the administration of sodium bicarbonate to humans: arterial dilatation in response to carbon dioxide; decrease of the hematocrit, and intracellular cerebral acidosis. (author)

  14. Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

    International Nuclear Information System (INIS)

    Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

    1987-01-01

    We studied total cell-associated A14-[ 125 I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[ 125 I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[ 125 I]insulin, [ 125 I]iodide, or [ 125 I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients

  15. Simple Recovery of Intracellular Gold Nanoparticles from Peanut Seedling Roots.

    Science.gov (United States)

    Raju, D; Mehta, Urmil J; Ahmad, Absar

    2015-02-01

    Fabrication of inorganic nanomaterials via a biological route witnesses the formation either extracellularly, intracellulary or both. Whereas extracellular formation of these nanomaterials is cherished owing to their easy and economical extraction and purification processes; the intracellular formation of nanomaterials, due to the lack of a proper recovery protocol has always been dreaded, as the extraction processes used so far were tedious, costly, time consuming and often resulting in very low recovery. The aim of the present study was to overcome the problems related with the extraction and recovery of intracellularly synthesized inorganic nanoparticles, and to devise a method to increasing the output, the shape, size, composition and dispersal of nanoparticles is not altered. Water proved to be much better system as it provided well dispersed, stable gold nanoparticles and higher recovery. This is the first report, where intracellular nanoparticles have been recovered using a very cost-effective and eco-friendly approach.

  16. Intracellular Ca2+ Regulation in Calcium Sensitive Phenotype of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    HERMANSYAH

    2010-03-01

    Full Text Available Intracellular cytosolic Ca2+ concentration accumulation plays an essential information in Saccharomyces cerevisiae i.e. to explain cellular mechanism of Ca2+ sensitive phenotype. Disruption both S. cerevisiae PPase PTP2 and MSG5 genes showed an inhibited growth in the presence of Ca2+. On the other hand, by using Luminocounter with apoaequorin system, a method based upon luminescent photoprotein aequorin, intracellular Ca2+ concentration was accumulated as a consequence of calcium sensitive phenotype of S. cerevisiae. This fact indicated that PPase ptp2Δ and msg5Δ were involved in intracellular Ca2+ transport in addition their already known pathways i.e Mitogen Activated Protein Kinase cell wall integrity pathway, high osmolarity glycerol (HOG pathway, and pheromone response FUS3 pathway.

  17. Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing1[C][W][OPEN

    Science.gov (United States)

    Zhang, Wei; Liu, Tianqi; Ren, Guodong; Hörtensteiner, Stefan; Zhou, Yongming; Cahoon, Edgar B.; Zhang, Chunyu

    2014-01-01

    Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis. PMID:25059706

  18. Mycobacterium avium-intracellulare cellulitis occurring with septic arthritis after joint injection: a case report

    Directory of Open Access Journals (Sweden)

    Murdoch David M

    2007-02-01

    Full Text Available Abstract Background Cellulitis caused by Mycobacterium avium-intracellulare has rarely been described. Mycobacterium avium-intracellulare is a rare cause of septic arthritis after intra-articular injection, though the causative role of injection is difficult to ascertain in such cases. Case presentation A 57-year-old with rheumatoid arthritis treated with prednisone and azathioprine developed bilateral painful degenerative shoulder arthritis. After corticosteroid injections into both acromioclavicular joints, he developed bilateral cellulitis centered over the injection sites. Skin biopsy showed non-caseating granulomas, and culture grew Mycobacterium avium-intracellulare. Joint aspiration also revealed Mycobacterium avium-intracellulare infection. Conclusion Although rare, skin and joint infections caused by Mycobacterium avium-intracellulare should be considered in any immunocompromised host, particularly after intra-articular injection. Stains for acid-fast bacilli may be negative in pathologic samples even in the presence of infection; cultures of tissue specimens should always be obtained.

  19. Identification and characterization of carprofen as a multitarget fatty acid amide hydrolase/cyclooxygenase inhibitor.

    Science.gov (United States)

    Favia, Angelo D; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2012-10-25

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the nonsteroidal anti-inflammatory drug carprofen as a multitarget-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2, and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several derivatives of carprofen, sharing this multitarget activity. This may result in improved analgesic efficacy and reduced side effects (Naidu et al. J. Pharmacol. Exp. Ther.2009, 329, 48-56; Fowler, C. J.; et al. J. Enzyme Inhib. Med. Chem.2012, in press; Sasso et al. Pharmacol. Res.2012, 65, 553). The new compounds are among the most potent multitarget FAAH/COX inhibitors reported so far in the literature and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs.

  20. Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates

    DEFF Research Database (Denmark)

    Kamp, Anja; Stief, Peter; Bristow, Laura A.

    2016-01-01

    % was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before...... store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom......-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly...

  1. 1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

    DEFF Research Database (Denmark)

    Troelsen, J T; Mehlum, A; Spodsberg, N

    1994-01-01

    Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity...... to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due...

  2. Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates

    Directory of Open Access Journals (Sweden)

    Anja Kamp

    2016-11-01

    Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

  3. Two sides of the same coin: Xyloglucan endotransglucosylases/hydrolases in host infection by the parasitic plant Cuscuta.

    Science.gov (United States)

    Olsen, Stian; Popper, Zoë A; Krause, Kirsten

    2016-01-01

    The holoparasitic angiosperm Cuscuta develops haustoria that enable it to feed on other plants. Recent findings corroborate the long-standing theory that cell wall modifications are required in order for the parasite to successfully infect a host, and further suggest that changes to xyloglucan through the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) are essential. On the other hand, XTH expression was also detected in resistant tomato upon an attack by Cuscuta, which suggests that both host and parasite use these enzymes in their "arms race." Here, we summarize existing data on the cell wall-modifying activities of XTHs during parasitization and present a model suggesting how XTHs might function to make the host's resources accessible to Cuscuta.

  4. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

    Directory of Open Access Journals (Sweden)

    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  5. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    International Nuclear Information System (INIS)

    Majumdar, S.; Basu, S.K.

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

  6. Complexation of intracellular cyanide by hydroxocobalamin using a human cellular model.

    Science.gov (United States)

    Astier, A; Baud, F J

    1996-01-01

    1. The rational for administering hydroxocobalamin (OHCbl) as an antidote to cyanide poisoning is based on the high affinity of CN ion for cobalt compounds. However, only few data are available on the influence of OHCbl on the intracellular cyanide pool. 2. In human fibroblasts incubated for 10 min with 500 microM of [14C] cyanide, the accumulation ratio was 25 at 37 degrees C (10.45 +/- 1.51 mM) and 11.9 at 4 degrees C. 3. Using the monoblastic U-937 cell line, a rapid uptake of radioactive cyanide was observed with a maximum accumulation ratio of 1.97 at 5 min. 4. A linear relationship between cyanide uptake by U-937 cells and cyanide concentration in incubation medium (10-500 microM; 5 min) was found suggesting a first order process (k = 0.25 min-1). 5. After incubation of fibroblasts with 500 microM of OHCbl, a 75% decrease of intracellular cyanide was observed, with concomittant formation of intracellular cyanocobalamin CNCbl (intracellular/extracellular ratio: 158). 6. These findings suggest that OHCbl is able to penetrate into heavily cyanide loaded cells and to complex cyanide to the non-toxic CNCbl form.

  7. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil

    Directory of Open Access Journals (Sweden)

    Ricardo Rodrigues de Melo

    Full Text Available ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

  8. Dynamics of gradient formation by intracellular shuttling

    Energy Technology Data Exchange (ETDEWEB)

    Berezhkovskii, Alexander M. [Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 (United States); Shvartsman, Stanislav Y. [Department of Chemical and Biological Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544 (United States)

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  9. Electron Microscopy of Intracellular Protozoa

    Science.gov (United States)

    1988-12-20

    Classification) " ELECTRON MICROSCOPY OF INTRACELLULAR PROTOZOA 12. PERSONAL AUTHOR(S) Aikawa, Masamichi 13a. TYPE OF REPORT I13b. TIME COVERED 114...authors suggest that anti-CS protein antibody is important in reducing the prevalence of malaria with increasing age among persons in such areas and... Hygine 33, 220-226. 0Giudice, G.D., Engers, H.D., Tougne, C., Biro, S.S., Weiss, N., Verdini, A.S., Pessi, A., Degremont, A.A., Freyvogel, T.A., Lambert

  10. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

    Science.gov (United States)

    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

    Science.gov (United States)

    Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

    2016-01-01

    To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

  12. Development and Properties of a Wax Ester Hydrolase in the Cotyledons of Jojoba Seedlings 1

    Science.gov (United States)

    Huang, Anthony H. C.; Moreau, Robert A.; Liu, Kitty D. F.

    1978-01-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent Km value for N-methylindoxylmyristate was 93 μM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID:16660288

  13. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Directory of Open Access Journals (Sweden)

    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  14. Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides

    Science.gov (United States)

    2004-01-01

    XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of α-D-galacturonic acid, highly substituted with β-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were derived from an XGH digestion of gum tragacanth, then fractionated, and analysed for their sugar composition and structure by matrix-assisted laser-desorption ionization–time-of-flight MS and nanospray MS. Several oligosaccharides from XGA were identified with different galacturonic acid/xylose ratios including five oligosaccharide isomers. Although XGH can act as an endo-enzyme, product-progression profiling showed that the disaccharide GalAXyl was predominantly produced from XGA by XGH, which indicated also an exolytic action. The latter was further supported by degradation studies of purified oligosaccharide GalA4Xyl3. It was shown that XGH acted from the non-reducing end towards the reducing end of this oligosaccharide, and showed the processive character of XGH. The results from this study further show that although XGH prefers to act between two xylosidated GalA units, it tolerates unsubstituted GalA units in its −1 and +1 subsites. PMID:15560751

  15. DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

  16. Intracellular pH and inorganic phosphate content of heart in vivo: A 31P-NMR study

    International Nuclear Information System (INIS)

    Katz, L.A.; Swain, J.A.; Portman, M.A.; Balaban, R.S.

    1988-01-01

    Studies were performed to determine the contribution of red blood cells to the 31 P-nuclear magnetic resonance (NMR) spectrum of the canine heart in vivo and the feasibility of measuring myocardial intracellular phosphate and pH. This was accomplished by replacing whole blood with a perfluorochemical perfusion emulsion blood substitute, Oxypherol, and noting the difference in the 31 P-NMR spectrum of the heart. NMR data were collected with a NMR transmitter-receiver coil on the surface of the distal portion of the left ventricle. These studies demonstrated that a small contribution from 2,3-diphosphoglycerate (2,3-DPG) and phosphodiesters in the blood could be detected. The magnitude and shift of these blood-borne signals permitted the relative quantification of intracellular inorganic phosphate (P i ) content as well as intracellular pH. Under resting conditions, the intracellular ATP/P i was 7.0 ± 0.08. This corresponds to a free intracellular P 1 content of ∼ 0.8 μmol./g wet wt. The intracellular pH was 7.10 ± 0.01. Acute respiratory alkalosis and acidosis, with the arterial pH ranging from ∼7.0 to 7.7, resulted in only small changes in the intracellular pH. These latter results demonstrate an effective myocardial intracellular proton-buffering mechanism in vivo

  17. Chelation of intracellular calcium blocks insulin action in the adipocyte

    International Nuclear Information System (INIS)

    Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

    1987-01-01

    The hypothesis that intracellular Ca 2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca 2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl 2 and the Ca 2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl 2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 μM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125 I=labeled insulin to adipocytes. These findings suggest that intracellular Ca 2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca 2+ -dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin

  18. Effect of insulin resistance on intracellular signal transduction of vessels in diabetic

    International Nuclear Information System (INIS)

    Cen Rongguang; Wei Shaoying; Mo Xingju

    2003-01-01

    To investigate the relationship between the insulin resistance (IR) and the intracellular signal transduction of vessels, changes in fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), inositol triphosphate (IP 3 ), protein kinase C(PKC) and intracellular total calcium concentration in 31 diabetic patients were compared with those of 39 normal controls. The levels of FBG, FINS, TG and TC in diabetic patients were significantly higher than those of normal controls (P 3 and PKC in diabetic patients were significantly lower than those of normal controls (P<0.01). The results suggest that there is a causal relation between insulin resistance and abnormalities of cellular calcium metabolism and intracellular signal transduction of vessels

  19. Crystal Structure of Homoserine Transacetylase from Haemophilus Influenzae Reveals a New Family of alpha/beta-Hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Mirza,I.; Nazi, I.; Korczynska, M.; Wright, G.; Berghuis, A.

    2005-01-01

    Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the properties of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs. hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.

  20. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

    Science.gov (United States)

    Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

    2016-04-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

  1. Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

    Energy Technology Data Exchange (ETDEWEB)

    Karpus, Olga N.; Hsiao, Cheng-Chih; Kort, Hanneke de; Tak, Paul P.; Hamann, Jörg, E-mail: j.hamann@amc.uva.nl

    2016-08-26

    Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS. - Highlights: • Intracellular poly(I:C) and 3pRNA evoke immune responses in FLS. • Only intracellular delivery of poly(I:C) induces FLS apoptosis. • FLS do not require MDA5 for their response to intracellular poly(I:C). • FLS respond to intracellular poly(I:C) independent of IPS and STING. • An unknown intracellular sensor recognizes linear dsRNA in FLS.

  2. Intracellular pH gradients in migrating cells

    DEFF Research Database (Denmark)

    Martin, Christine; Pedersen, Stine Helene Falsig; Schwab, Albrecht

    2011-01-01

    might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of p...

  3. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent. Production of nanoparticles was confirmed by the colour ...

  4. Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

    Science.gov (United States)

    Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

    2018-02-05

    During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

  5. Purification and Characterization of Tannin Acyl Hydrolase from Aspergillus niger ATCC 16620

    Directory of Open Access Journals (Sweden)

    Abdulhameed Sabu

    2005-01-01

    Full Text Available Tannin acyl hydrolase produced extracellularly by the fungal strain Aspergillus niger ATTC 16620 in solid state fermentation was purified from the cell free culture broth by ammonium sulphate fractionation followed by DEAE–Sephadex A-50 chromatography. SDS-PAGE analysis indicated that the enzyme protein molecular mass was 168 kDa. Enzyme activity was stable up to the temperature of 40 °C and the enzyme activity was optimal at pH=6. Tannase activity was maximal at 0.01 M concentration of the substrate. The addition of metal ions like Zn2+, Mn2+, Cu2+, Ca2+, Mg2+and Fe2+ inhibited the enzyme activity. Only K+ ions enhanced tannase activity, and an activity of 4.31 U/mL was reported here. Enzyme activity was maximal after 15–20 min of incubation time, with an activity of 3.9 U/mL. Km was found to be 1.03 mM and Vmax=4.25 mmol/min. Since the enzyme is active over a wide range of pH and temperature it could find potential use in the food-processing industry.

  6. The Structural Basis of Exopolygalacturonase Activity in a Family 28 Glycoside Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Abbott,D.; Boraston, A.

    2007-01-01

    Family 28 glycoside hydrolases (polygalacturonases) are found in organisms across the plant, fungal and bacterial kingdoms, where they are central to diverse biological functions such as fruit ripening, biomass recycling and plant pathogenesis. The structures of several polygalacturonases have been reported; however, all of these enzymes utilize an endo-mode of digestion, which generates a spectrum of oligosaccharide products with varying degrees of polymerization. The structure of a complementary exo-acting polygalacturonase and an accompanying explanation of the molecular determinants for its specialized activity have been noticeably lacking. We present the structure of an exopolygalacturonase from Yersinia enterocolitica, YeGH28 in a native form (solved to 2.19 {angstrom} resolution) and a digalacturonic acid product complex (solved to 2.10 {angstrom} resolution). The activity of YeGH28 is due to inserted stretches of amino acid residues that transform the active site from the open-ended channel observed in the endopolygalacturonases to a closed pocket that restricts the enzyme to the exclusive attack of the non-reducing end of oligogalacturonide substrates. In addition, YeGH28 possesses a fused FN3 domain with unknown function, the first such structure described in pectin active enzymes.

  7. Pulsed magneto-motive ultrasound imaging to detect intracellular accumulation of magnetic nanoparticles

    International Nuclear Information System (INIS)

    Mehrmohammadi, Mohammad; Qu Min; Sokolov, Konstantin V; Emelianov, Stanislav Y; Ma, Li L; Johnston, Keith P; Romanovicz, Dwight K

    2011-01-01

    As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular accumulation of nanoparticles-an important part of cell-nanoparticle interaction-has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique-pulsed magneto-motive ultrasound (pMMUS)-to identify intracellular accumulation of endocytosed magnetic nanoparticles. In pMMUS imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to the signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular accumulation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular accumulation non-invasively and in real-time.

  8. Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2.

    Directory of Open Access Journals (Sweden)

    David Talens-Perales

    Full Text Available In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2. We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module with two β-sandwich domains (non-catalytic at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

  9. [Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

    Science.gov (United States)

    Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

    2014-03-01

    In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

  10. Crystal Structure of Homo Sapiens PTD012 Reveals a Zinc-Containing Hydrolase Fold

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty,B.; Bussow, K.; Fieber-ErdMan, M.; Roske, Y.; Gobam, J.; Scheich, C.; Gotz, F.; Niesen, F.; Heinemann, U.

    2006-01-01

    The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 Angstroms resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an {alpha}{beta}{beta}{alpha} four-layer topology. A metal ion residing between the central {beta}-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn{sup 2+}. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn{sup 2+} to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and {beta}-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.

  11. Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

    Science.gov (United States)

    Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

    2004-05-15

    Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

  12. Getting “Inside” Type I IFNs: Type I IFNs in Intracellular Bacterial Infections

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    Deann T. Snyder

    2017-01-01

    Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

  13. Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy.

    Science.gov (United States)

    Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

    2013-03-01

    Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellulare pulmonary disease, the patient was clinically well at a 1-month follow-up.

  14. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

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    Onkar Sharma

    2016-03-01

    Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

  15. Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

    Science.gov (United States)

    Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

    2004-10-22

    (4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu

  16. Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

    OpenAIRE

    Pesce , S.; Beguet , J.; Rouard , N.; Devers Lamrani , M.; Martin Laurent , F.

    2013-01-01

    A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-14C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sedimen...

  17. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases.

    Science.gov (United States)

    Calderan-Rodrigues, Maria Juliana; Jamet, Elisabeth; Douché, Thibaut; Bonassi, Maria Beatriz Rodrigues; Cataldi, Thaís Regiani; Fonseca, Juliana Guimarães; San Clemente, Hélène; Pont-Lezica, Rafael; Labate, Carlos Alberto

    2016-01-11

    Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane

  19. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

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    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  20. Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

    Science.gov (United States)

    Kang, Yoon-Suk; Kirby, James E

    2017-05-01

    We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

  1. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

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    Brinton Roberta

    2008-12-01

    Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that AP

  2. Intracellular Bacterial Infections: A Challenge for Developing Cellular Mediated Immunity Vaccines for Farmed Fish

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    Hetron Mweemba Munang’andu

    2018-04-01

    Full Text Available Aquaculture is one of the most rapidly expanding farming systems in the world. Its rapid expansion has brought with it several pathogens infecting different fish species. As a result, there has been a corresponding expansion in vaccine development to cope with the increasing number of infectious diseases in aquaculture. The success of vaccine development for bacterial diseases in aquaculture is largely attributed to empirical vaccine designs based on inactivation of whole cell (WCI bacteria vaccines. However, an upcoming challenge in vaccine design is the increase of intracellular bacterial pathogens that are not responsive to WCI vaccines. Intracellular bacterial vaccines evoke cellular mediated immune (CMI responses that “kill” and eliminate infected cells, unlike WCI vaccines that induce humoral immune responses whose protective mechanism is neutralization of extracellular replicating pathogens by antibodies. In this synopsis, I provide an overview of the intracellular bacterial pathogens infecting different fish species in aquaculture, outlining their mechanisms of invasion, replication, and survival intracellularly based on existing data. I also bring into perspective the current state of CMI understanding in fish together with its potential application in vaccine development. Further, I highlight the immunological pitfalls that have derailed our ability to produce protective vaccines against intracellular pathogens for finfish. Overall, the synopsis put forth herein advocates for a shift in vaccine design to include CMI-based vaccines against intracellular pathogens currently adversely affecting the aquaculture industry.

  3. Semiconductor quantum dots as Förster resonance energy transfer donors for intracellularly-based biosensors

    Science.gov (United States)

    Field, Lauren D.; Walper, Scott A.; Susumu, Kimihiro; Oh, Eunkeu; Medintz, Igor L.; Delehanty, James B.

    2017-02-01

    Förster resonance energy transfer (FRET)-based assemblies currently comprise a significant portion of intracellularly based sensors. Although extremely useful, the fluorescent protein pairs typically utilized in such sensors are still plagued by many photophysical issues including significant direct acceptor excitation, small changes in FRET efficiency, and limited photostability. Luminescent semiconductor nanocrystals or quantum dots (QDs) are characterized by many unique optical properties including size-tunable photoluminescence, broad excitation profiles coupled to narrow emission profiles, and resistance to photobleaching, which can cumulatively overcome many of the issues associated with use of fluorescent protein FRET donors. Utilizing QDs for intracellular FRET-based sensing still requires significant development in many areas including materials optimization, bioconjugation, cellular delivery and assay design and implementation. We are currently developing several QD-based FRET sensors for various intracellular applications. These include sensors targeting intracellular proteolytic activity along with those based on theranostic nanodevices for monitoring drug release. The protease sensor is based on a unique design where an intracellularly expressed fluorescent acceptor protein substrate assembles onto a QD donor following microinjection, forming an active complex that can be monitored in live cells over time. In the theranostic configuration, the QD is conjugated to a carrier protein-drug analogue complex to visualize real-time intracellular release of the drug from its carrier in response to an external stimulus. The focus of this talk will be on the design, properties, photophysical characterization and cellular application of these sensor constructs.

  4. The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S; Vafiadi, Christina; Dijkhuizen, Lubbert

    BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the

  5. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    Science.gov (United States)

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  6. Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

    Science.gov (United States)

    Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

    2018-06-11

    Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

    Directory of Open Access Journals (Sweden)

    Sha Liu

    Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

  8. Intrinsically disordered regions may lower the hydration free energy in proteins: a case study of nudix hydrolase in the bacterium Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Omar Awile

    Full Text Available The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the "surface-properties" of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity.

  9. Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

    Science.gov (United States)

    Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

    2012-01-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies. PMID:22851196

  10. Intracellular bacteria: the origin of dinoflagellate toxicity.

    Science.gov (United States)

    Silva, E S

    1990-01-01

    Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

  11. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

    International Nuclear Information System (INIS)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; Trindade da Silva, Carlos Antonio; Morisseau, Christophe; Hammock, Bruce D.

    2015-01-01

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE 2 was monitored. While OME treatment by itself exhibited variable effects on PGE 2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  12. Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems.

    Science.gov (United States)

    Ngo, Anh H; Bose, Sohini; Do, Loi H

    2018-03-23

    This concept article focuses on the rapid growth of intracellular chemistry dedicated to the integration of small-molecule metal catalysts with living cells and organisms. Although biological systems contain a plethora of biomolecules that can deactivate inorganic species, researchers have shown that small-molecule metal catalysts could be engineered to operate in heterogeneous aqueous environments. Synthetic intracellular reactions have recently been reported for olefin hydrogenation, hydrolysis/oxidative cleavage, azide-alkyne cycloaddition, allylcarbamate cleavage, C-C bond cross coupling, and transfer hydrogenation. Other promising targets for new biocompatible reaction discovery will also be discussed, with a special emphasis on how such innovations could lead to the development of novel technologies and chemical tools. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

    Science.gov (United States)

    Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

    2015-12-01

    A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

  14. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  15. Regulation of dopamine transporter trafficking by intracellular amphetamine

    DEFF Research Database (Denmark)

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang

    2006-01-01

    -induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent...... alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic...... redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A...

  16. Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

    Science.gov (United States)

    Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

    2014-01-01

    Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

  17. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shangrong; Wang, Yifan; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

    2014-06-13

    Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

  18. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

    International Nuclear Information System (INIS)

    Zhang, Shangrong; Wang, Yifan; Li, Shu Jie

    2014-01-01

    Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor

  19. Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

    Directory of Open Access Journals (Sweden)

    Olga Maslova

    2017-09-01

    Full Text Available Catalytic characteristics of hexahistidine-containing organophosphorus hydrolase (His6-OPH and its enzyme-polyelectrolyte complexes with poly-l-glutamic acid or poly-l-aspartic acid (His6-OPH/PLD50, hydrolyzing organophosphorous compounds, and N-acyl homoserine lactones were studied in the presence of various antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin. The antibiotics at concentrations below 1 g·L−1 had a negligible inhibiting effect on the His6-OPH activity. Mixed inhibition of His6-OPH was established for higher antibiotic concentrations, and rifampicin was the most potent inhibitor. Stabilization of the His6-OPH activity was observed in the presence of antibiotics at a concentration of 0.2 g·L−1 during exposure at 25–41 °C. Molecular docking of antibiotics to the surface of His6-OPH dimer revealed the antibiotics binding both to the area near active centers of the enzyme subunits and to the region of contact between subunits of the dimer. Such interactions between antibiotics and His6-OPH were verified with Fourier-transform infrared (FTIR spectroscopy. Considering all the results of the study, the combination of His6-OPH/PLD50 with β-lactam antibiotic ampicillin was established as the optimal one in terms of exhibition and persistence of maximal lactonase activity of the enzyme.

  20. New insights into plant glycoside hydrolase family 32 in Agave species.

    Science.gov (United States)

    Avila de Dios, Emmanuel; Gomez Vargas, Alan D; Damián Santos, Maura L; Simpson, June

    2015-01-01

    In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  1. Biological macromolecules based targeted nanodrug delivery systems for the treatment of intracellular infections.

    Science.gov (United States)

    Aparna, V; Shiva, M; Biswas, Raja; Jayakumar, R

    2018-04-15

    Intracellular infections are tricky to treat, the reason being the poor penetration of antibiotics/antimycotics into the microbial niche (host cell). Macrophages are primary targets of facultative and obligate intracellular bacteria/fungi to be abused as host cells. The need for drugs with better intracellular penetration led to the development of endocytosable drug carriers, which can cross the cell membrane of the host cells (macrophages) by imitating the entry path of the pathogens. Therefore, the drugs can be targeted to macrophages ensuring enhanced therapeutic effect. This review discusses the exploitation of various nanocarriers for targeted delivery of drugs to the macrophages in the last two decades. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Estimating the biophysical properties of neurons with intracellular calcium dynamics.

    Science.gov (United States)

    Ye, Jingxin; Rozdeba, Paul J; Morone, Uriel I; Daou, Arij; Abarbanel, Henry D I

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V(t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  3. Azithromycin effectiveness against intracellular infections of Francisella

    Directory of Open Access Journals (Sweden)

    Mann Barbara J

    2010-04-01

    Full Text Available Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F. tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS (Type B was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA were found to be less sensitive in vitro to Az compared to the wild

  4. Study on the inactivation of intracellular enzyme molecules by X-ray irradiation

    International Nuclear Information System (INIS)

    Lee, S.B.

    1977-01-01

    Inactivation of the glutamic acid dehydrogenase and glucose-6-phosphate dehydrogenase enzyme molecules in the Ehrlich ascites tumor cells of the mouse were studied. The above mentioned intracellular enzyme molecules were irradiated by the X-ray radiation under the condition of 65 kV, 1 Amp under the atmosphere of nitrogen gases and by 4 0 C. Thereby, irradiation doses were 580 KR/min(error: +-3%). After irradiation, the cell homogentes were prepared through liquid air techniques. There after, the activities of the enzymes were measured with photometric method given by O. Warburg and W. Christian. The dose effect curves of the activities of the two enzymes by the X-ray irradiation showed both exponential and the inactivation doses were 6.5x10 6 and 5.0x10 6 R respectively. These results showed one side that the inactivation process of the intracellular enzyme molecules was one hit reaction after target theory, and the other side that this inactivation process could not be the primary causes of the death through X-ray irradiation of the vertebrate animals, because of the high resistance of the intracellular protein molecules against X-ray irradiation. The one hit reaction by the inactivation process of the irradiated intracellular enzyme molecules was discussed. (author)

  5. Intracellular pH-sensing using core/shell silica nanoparticles.

    Science.gov (United States)

    Korzeniowska, B; Woolley, R; DeCourcey, J; Wencel, D; Loscher, C E; McDonagh, C

    2014-07-01

    An in-depth understanding of biochemical processes occurring within biological systems is key for early diagnosis of disease and identification of appropriate treatments. Nanobiophotonics offers huge potential benefits for intracellular diagnostics and therapeutics. Intracellular sensing using fluorescent nanoparticles is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of optical chemical nanosensors for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly silica matrix with co-encapsulated Texas Red, acting as a reference dye, and pH-sensitive fluorescein isothiocyanate enabling ratiometric quantitative environmental detection. In order to obtain silica-based nanoparticles -70 nm in size, a modified sol-gel-based Stöber method was employed. The potential of these nanosensors for intracellular pH monitoring is demonstrated inside a live human embryonic kidney cell line whereby a significant change in fluorescence is observed when the cell pH is switched from acidic to basic. High loading efficiencies of nanoparticles into the cells is seen, with little effect on cell morphology even following extended nanoparticle exposure (up to 72 h). Nanoparticle incubation time and the fast response of the nanosensor (-2 s) make it a very powerful tool in monitoring the processes occurring within the cytosol.

  6. Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

    Science.gov (United States)

    Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan

    2016-10-03

    The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

  7. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    Science.gov (United States)

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  8. Salmonella Intracellular Lifestyles and Their Impact on Host-to-Host Transmission.

    Science.gov (United States)

    Pucciarelli, M Graciela; García-Del Portillo, Francisco

    2017-07-01

    More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella -containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.

  9. Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

    Science.gov (United States)

    Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2013-01-01

    Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328

  10. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil.

    Science.gov (United States)

    Melo, Ricardo Rodrigues de; Persinoti, Gabriela Felix; Paixão, Douglas Antonio Alvaredo; Squina, Fábio Márcio; Ruller, Roberto; Sato, Helia Harumi

    Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296bp and G+C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  11. Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

    Directory of Open Access Journals (Sweden)

    Dougherty Michael J

    2012-07-01

    Full Text Available Abstract Background Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results Twenty-two putative ORFs (open reading frames were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.

  12. Effects of Cu, Zn and Pb Combined Pollution on Soil Hydrolase Activities

    Directory of Open Access Journals (Sweden)

    FENG Dan

    2015-08-01

    Full Text Available To study the relations between soil enzyme activities and heavy metal pollution, the combined effects of Cu, Zn and Pb on the three hydrolase activities, including invertase(IN, urease(Uand alkaline phosphatase(ALPwere investigated via an orthogonal experiment. Results showed as the following: When the concentration of Cu was 400 mg·kg-1, the U and ALP activities were decreased 51% and 44%, separately; When Zn was at 500 mg·kg-1, IN and ALP activities were only decreased 3% and 9%, while U activity was increased; When Pb was at 500 mg·kg-1, IN and U activities were increased, while ALP activity was decreased 13%. As a whole, Cu was considered as the most remarkable influence factor for IN, U and ALP activity regardless of interactions among the heavy metals, Zn came second, and Pb mainly showed activation. Considering interactions, Cu×Zn could significantly influence U activity(P<0.05, effects of Cu×Pb and Cu×Zn on ALP activity were remarkable(95% confidence interval. The response of ALP activity was more sensitive than the other two enzymes. Soil ALP activity might be a sensitive tool for assessing the pollution degree of Cu.

  13. Intracellular serpins, firewalls and tissue necrosis.

    Science.gov (United States)

    Marciniak, Stefan J; Lomas, David A

    2008-02-01

    Luke and colleagues have recently attributed a new role to a member of the serpin superfamily of serine proteinase inhibitors. They have used Caenorhabditis elegans to show that an intracellular serpin is crucial for maintaining lysosomal integrity. We examine the role of this firewall in preventing necrosis and attempt to integrate this with current theories of stress-induced protein degradation. We discuss how mutant serpins cause disease either through polymerization or now, perhaps, by unleashing necrosis.

  14. Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

    OpenAIRE

    Yadav, Kavita; Kumar, Chanchal; Archana, G.; Naresh Kumar, G.

    2014-01-01

    Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) s...

  15. Cytoplasmic tail of coronavirus spike protein has intracellular

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

  16. Facilitating Intracellular Drug Delivery by Ultrasound-Activated Microbubbles

    NARCIS (Netherlands)

    Lammertink, BHA

    2017-01-01

    The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model

  17. Heavy metals toxicity after acute exposure of cultured renal cells. Intracellular accumulation and repartition

    International Nuclear Information System (INIS)

    Khodja, Hicham; Carriere, Marie; Avoscan, Laure; Gouget, Barbara

    2005-01-01

    Lead (Pb), cadmium (Cd) and uranium (U) present no known biological function but are toxic in various concentration ranges. Pb and Cd lead generally to nephrotoxicity consisting in proximal renal tubular dysfunction and accumulation while U has been reported to induce chemical kidney toxicity, functional and histological damages being as well mainly observed in proximal tubule cells. This work address the question of Cd, Pb, and U cytotoxicity, intracellular accumulation and repartition after acute intoxication of renal proximal tubule epithelial cells. After cells exposure to different concentrations of metals for various times, morphological changes were observed and intracellular concentrations and distributions of toxic metals were specified by PIXE coupled to RBS. Cell viability, measured by biochemical tests, was used as toxicity indicator. A direct correlation between cytotoxicity and intracellular accumulation in renal epithelial cells have been established. Finally, intracellular Pb and U localizations were detected while Cd was found to be uniformly distributed in renal cells. (author)

  18. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  19. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine

    Directory of Open Access Journals (Sweden)

    N. V. Olkhovych

    2016-10-01

    Full Text Available The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W and c.745C>T (R249W in the HEXA gene, c.1726G>A (G576S and c.2065G>A (E689K in the GAA gene, c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease. The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes was 10.3%. The frequency of c.739C>T (R247W allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of

  20. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine.

    Science.gov (United States)

    Olkhovych, N V; Gorovenko, N G

    2016-01-01

    The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may

  1. Ubiquitin Carboxy-Terminal HydrolaseL3 Correlates with Human Sperm Count, Motility and Fertilization.

    Science.gov (United States)

    Wang, Meijiao; Yu, Tinghe; Hu, Lina; Cheng, Zhi; Li, Min

    2016-01-01

    Ubiquitin C-terminal hydrolase L3 (UCHL3) belongs to the group of deubiquitinating enzymes and plays a part in apoptosis of germ cells and the differentiation of spermatocytes into spermatids. However, the exact role of UCHL3 in human spermatogenesis and sperm function remains unknown. Here we examined the level and activity of UCHL3 in spermatozoa from men with asthenozoospermia (A), oligoasthenozoospermia (OA) or normozoospermia (N). Immunofluorescence indicated that UCHL3 was mainly localized in the acrosome and throughout the flagella, and western blotting revealed a lower level in A or OA compared with N (p sperm count, concentration and motility. The UCHL3 level was positively correlated with the normal fertilization rate (FR) and percentage of embryos suitable for transfer/cryopreservation of in vitro fertilization (IVF). The UCHL3 activity was also positively correlated with FR, the percentage of embryos suitable for transfer/cryopreservation and high-quality embryos rate of IVF. Aforementioned correlations were not manifested in intra-cytoplasmic sperm injection (ICSI). These findings suggest that UCHL3 may play a role in male infertility.

  2. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    Science.gov (United States)

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-05-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where particles are abundantly internalized - is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a ``lysosome-enhanced Trojan horse effect'' since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where

  3. Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ming-Ju Chou

    2018-02-01

    Full Text Available Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

  4. Mechanisms of cellular invasion by intracellular parasites.

    Science.gov (United States)

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  5. Intracellular thiol levels and radioresistance: Studies with glutathione and glutathione mono ethyl ester

    International Nuclear Information System (INIS)

    Astor, M.B.; Meister, A.; Anderson, M.E.

    1987-01-01

    Intracellular thiols such as glutathione (GSH) protect cells against free radicals formed during oxidative metabolism or from exposure to drugs or ionizing radiation. The role of intracellular GSH in the repair of radiation induced free radical damage was studied using GSH or its analog glutathione mono ethyl ester (GEE), which readily penetrates into the cell. Chinese hamster V79 cells with normal GSH levels were afforded equal protection under aerated and hypoxic conditions (DMF = 1.2 OER = 3.7) by both 10 mM GSH and GEE although GEE had raised interacellular GSH levels three-fold. Growth of V79 cells in cysteine free media resulted in undetectable levels of GSH and OER of 2.2 with no change in aerated survival. Restoration of intracellular GSH by 10 mM GEE resulted in an increase of the OER from 2.2. to 3.8 (DMF = 1.7). Only 14% of the intracellular GSH needs to be repleted to give an OER of 3.0. These experiments provide evidence that thiols do play a role in the oxygen effect and are present at levels in excess of what is necessary for maximal radioprotection

  6. Conditions With High Intracellular Glucose Inhibit Sensing Through Glucose Sensor Snf3 in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Karhumaa, Kaisa; Wu, B.Q.; Kielland-Brandt, Morten

    2010-01-01

    as for amino acids. An alternating-access model of the function of transporter-like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose...... through the transporter-like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity...... of extracellular glucose to Snf3 was measured for cells grown in non-fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating-access model for transporter-like sensors. J. Cell. Biochem. 110: 920...

  7. Palatal Actinomycosis and Kaposi Sarcoma in an HIV-Infected Subject with Disseminated Mycobacterium avium-intracellulare Infection

    Directory of Open Access Journals (Sweden)

    Yuria Ablanedo-Terrazas

    2012-01-01

    Full Text Available Actinomyces and Mycobacterium avium-intracellulare are facultative intracellular organisms, members of the bacterial order actinomycetales. Although Actinomyces can behave as copathogen when anatomic barriers are compromised, its coinfection with Mycobacterium avium-intracellulare has not previously been reported. We present the first reported case of palatal actinomycosis co-infection with disseminated MAC, in an HIV-infected subject with Kaposi sarcoma and diabetes. We discuss the pathogenesis of the complex condition of this subject.

  8. Intracellular nitrate in sediments of an oxygen-deficient marine basin is linked to pelagic diatoms

    DEFF Research Database (Denmark)

    Kamp, Anja; Petro, Caitlin; Røy, Hans

    2018-01-01

    Intracellular nitrate is an important electron acceptor in oxygen-deficient aquatic environments, either for the nitrate-storing microbes themselves, or for ambient microbial communities through nitrate leakage. This study links the spatial distribution of intracellular nitrate with the abundance...... and identity of nitrate-storing microbes in sediments of the Bornholm Basin, an environmental showcase for severe hypoxia. Intracellular nitrate (up to 270 nmol cm−3 sediment) was detected at all 18 stations along a 35-km transect through the basin and typically extended as deep as 1.6 cm into the sediment...

  9. Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Aseem Pandey

    2018-04-01

    Full Text Available Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR sensor IRE1α (inositol-requiring enzyme 1 and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1 conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

  10. Intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory.

    Science.gov (United States)

    Takeda, Atsushi; Tamano, Haruna; Ogawa, Taisuke; Takada, Shunsuke; Nakamura, Masatoshi; Fujii, Hiroaki; Ando, Masaki

    2014-11-01

    The role of perforant pathway-dentate granule cell synapses in cognitive behavior was examined focusing on synaptic Zn(2+) signaling in the dentate gyrus. Object recognition memory was transiently impaired when extracellular Zn(2+) levels were decreased by injection of clioquinol and N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylendediamine. To pursue the effect of the loss and/or blockade of Zn(2+) signaling in dentate granule cells, ZnAF-2DA (100 pmol, 0.1 mM/1 µl), an intracellular Zn(2+) chelator, was locally injected into the dentate molecular layer of rats. ZnAF-2DA injection, which was estimated to chelate intracellular Zn(2+) signaling only in the dentate gyrus, affected object recognition memory 1 h after training without affecting intracellular Ca(2+) signaling in the dentate molecular layer. In vivo dentate gyrus long-term potentiation (LTP) was affected under the local perfusion of the recording region (the dentate granule cell layer) with 0.1 mM ZnAF-2DA, but not with 1-10 mM CaEDTA, an extracellular Zn(2+) chelator, suggesting that the blockade of intracellular Zn(2+) signaling in dentate granule cells affects dentate gyrus LTP. The present study demonstrates that intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory, probably via dentate gyrus LTP expression. Copyright © 2014 Wiley Periodicals, Inc.

  11. Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

    International Nuclear Information System (INIS)

    Phair, R.D.; Hai, C.M.

    1986-01-01

    A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

  12. Model-based control of the temporal patterns of intracellular signaling in silico

    Science.gov (United States)

    Murakami, Yohei; Koyama, Masanori; Oba, Shigeyuki; Kuroda, Shinya; Ishii, Shin

    2017-01-01

    The functions of intracellular signal transduction systems are determined by the temporal behavior of intracellular molecules and their interactions. Of the many dynamical properties of the system, the relationship between the dynamics of upstream molecules and downstream molecules is particularly important. A useful tool in understanding this relationship is a methodology to control the dynamics of intracellular molecules with an extracellular stimulus. However, this is a difficult task because the relationship between the levels of upstream molecules and those of downstream molecules is often not only stochastic, but also time-inhomogeneous, nonlinear, and not one-to-one. In this paper, we present an easy-to-implement model-based control method that makes the target downstream molecule to trace a desired time course by changing the concentration of a controllable upstream molecule. Our method uses predictions from Monte Carlo simulations of the model to decide the strength of the stimulus, while using a particle-based approach to make inferences regarding unobservable states. We applied our method to in silico control problems of insulin-dependent AKT pathway model and EGF-dependent Akt pathway model with system noise. We show that our method can robustly control the dynamics of the intracellular molecules against unknown system noise of various strengths, even in the absence of complete knowledge of the true model of the target system. PMID:28275530

  13. Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages.

    Directory of Open Access Journals (Sweden)

    M Azevedo

    Full Text Available The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.

  14. Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

    Science.gov (United States)

    Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

    2009-01-01

    BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

  15. Dihydroceramide biology - Structure-specific metabolism and intracellular localization

    NARCIS (Netherlands)

    Kok, JW; NikolovaKarakashian, M; Klappe, K; Alexander, C; Merrill, AH

    1997-01-01

    This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DN-Cer), an intermediate in de novo sphingolipid biosynthesis, When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl-DH-Cer (C-6-NBD-DH-Cer) was incubated with HT29, NRK, BHK,

  16. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE{sub 2} induced pain model

    Energy Technology Data Exchange (ETDEWEB)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D. [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Trindade da Silva, Carlos Antonio [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Department of Genetics and Biochemistry, Federal University of Uberlandia, MG (Brazil); Morisseau, Christophe [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States)

    2015-12-15

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE{sub 2} was monitored. While OME treatment by itself exhibited variable effects on PGE{sub 2} induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  17. Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

    Science.gov (United States)

    Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2017-03-28

    Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

  18. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements

    DEFF Research Database (Denmark)

    Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig

    2014-01-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells......, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped...... in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods....

  19. Clinical significance and epidemiologic analyses of Mycobacterium avium and Mycobacterium intracellulare lung disease from post-marketing surveillance.

    Science.gov (United States)

    Suzuki, Katsuhiro; Kurashima, Atsuyuki; Tatsuno, Kinji; Kadota, Jun-Ichi

    2018-01-01

    In Japan, nontuberculous mycobacterial lung disease is mostly attributable to Mycobacterium avium complex (MAC), i.e., M. avium or M. intracellulare. However, clinical features of the disease caused by these two pathogens have not been studied sufficiently yet. A post-marketing survey of clarithromycin was performed at 130 facilities across Japan. The data on patients with M. avium infection and patients with M. intracellulare infection were selected from this survey for comparison of background variables and clinical features of the two pathogens. Among the patients analyzed (n = 368), 67.4% had M. avium infection and 32.6% had M. intracellulare infection. Stratified analysis revealed no significant differences between the ratio of the two pathogens based on gender, disease type, complication, past medical history, or smoking history. However, the percentage of patients with M. intracellulare infection was significantly higher among those with underlying lung disease than among those without lung disease (p = 0.0217). The percentage of patients with M. intracellulare infection rose significantly with age (p = 0.0296). This age-related change was more significant in women (p = 0.0018). When district-wise analysis was performed for Japan, the percentage of M. intracellulare infection was higher in the Chugoku/Shikoku and Kyushu districts whereas the percentage of M. avium infection was higher in the other districts. This survey revealed some differences in the clinical and epidemiologic features of M. avium and M. intracellulare infection. The significant predominance of M. avium infection among relatively young women is suggestive of an increase in the M. avium/M. intracellulare infection ratio among women in the future. Copyright © 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

  20. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    Science.gov (United States)

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  1. Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

    Science.gov (United States)

    Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

    2016-06-01

    In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Bjerrum, Poul J; Jessen, Torben E

    2011-01-01

    BACKGROUND The vitamin D receptor (VDR) is expressed in human spermatozoa, and VDR-knockout mice and vitamin D (VD) deficiency in rodents results in impaired fertility, low sperm counts and a low number of motile spermatozoa. We investigated the role of activated VD (1,25(OH)(2)D(3)) in human...... spermatozoa and whether VD serum levels are associated with semen quality. METHODS Cross-sectional association study of semen quality and VD serum level in 300 men from the general population, and in vitro studies on spermatozoa from 40 men to investigate the effects of VD on intracellular calcium, sperm......M). 1,25(OH)(2)D(3) increased intracellular calcium concentration in human spermatozoa through VDR-mediated calcium release from an intracellular calcium storage, increased sperm motility and induced the acrosome reaction in vitro. CONCLUSIONS 1,25(OH)(2)D(3) increased intracellular calcium...

  3. New insights into plant glycoside hydrolase family 32 in Agave species

    Directory of Open Access Journals (Sweden)

    Emmanuel eAvila-de Dios

    2015-08-01

    Full Text Available In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (enzymes belonging to plant glycoside hydrolase family 32 from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae and A. striata. Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  4. Engineering of obligate intracellular bacteria: progress, challenges and paradigms

    Science.gov (United States)

    Over twenty years have passed since the first report of genetic manipulation of an obligate intracellular bacterium. Through progress interspersed by bouts of stagnation, microbiologists and geneticists have developed approaches to genetically manipulate obligates. A brief overview of the current ge...

  5. Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

    Science.gov (United States)

    Ludwar, Bjoern Ch; Evans, Colin G; Cropper, Elizabeth C

    2012-07-15

    It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record

  6. SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

    Science.gov (United States)

    Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

    2017-01-01

    We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

  7. Degradation of connective tissue matrices by macrophages. III. Morphological and biochemical studies on extracellular, pericellular, and intracellular events in matrix proteolysis by macrophages in culture

    International Nuclear Information System (INIS)

    Werb, Z.; Bainton, D.F.; Jones, P.A.

    1980-01-01

    The aim of the present study was to determine the localization of macrophage-mediated degradation of matrix proteins. The sites of matrix degradation were examined ultrastructurally, and the effects of modulation of macrophage secretion, endocytosis, and activity of macrophage hydrolases on matrix degradation were monitored biochemically

  8. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Directory of Open Access Journals (Sweden)

    Caroline da Silva Moraes

    2014-08-01

    Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

  9. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Science.gov (United States)

    Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

    2014-01-01

    The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

  10. Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

    Science.gov (United States)

    Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Valius, Mindaugas; Rotomskis, Ricardas

    2013-01-01

    Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison

  11. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  12. Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

  13. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    Science.gov (United States)

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  14. Gold-carbon dots for the intracellular imaging of cancer-derived exosomes

    Science.gov (United States)

    Jiang, Xiaoyue; Zong, Shenfei; Chen, Chen; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

    2018-04-01

    As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

  15. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

    Science.gov (United States)

    Knapp, W; Strobl, H; Majdic, O

    1994-12-15

    New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

  16. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Science.gov (United States)

    Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

    2013-01-01

    Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  17. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Directory of Open Access Journals (Sweden)

    Jinli Wang

    Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  18. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Science.gov (United States)

    Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

    2011-01-01

    Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  19. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Directory of Open Access Journals (Sweden)

    Yu-Hsuan Tu

    Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  20. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Directory of Open Access Journals (Sweden)

    Yves Briers

    Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  1. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Science.gov (United States)

    Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

    2012-01-01

    Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  2. Measurement of Intracellular Ionized Calcium in a Free-living Soil Nematode, Caenorhabditis elegans.

    Science.gov (United States)

    Kawaii, S; Yoshizawa, Y; Mizutani, J

    1993-01-01

    A calcium chelating fluorescence indicator, fura-2, was used to measure intracellular ionized calcium in Caenorhabditis elegans. The indicator loading process was harmless to the nematode, and completed within 2-3 h. Fura-2 was loaded mainly at its intestinal tract. The effects of DOPA on locomotion and the level of intracellular calcium were investigated and measured by using a microfluorometer. The addition of DOPA temporarily increased [Ca(2+)]i for several minutes.

  3. Fluorescent nanoparticles for intracellular sensing: a review.

    Science.gov (United States)

    Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

    2012-11-02

    Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Antimycobacterial Efficacy of Andrographis paniculata Leaf Extracts Under Intracellular and Hypoxic Conditions.

    Science.gov (United States)

    Bhatter, Purva; Gupta, Pooja; Daswani, Poonam; Tetali, Pundarikakshudu; Birdi, Tannaz

    2015-01-01

    The inhibition of the growth of Mycobacterium tuberculosis by the extracts of Andrographis paniculata has been studied using intracellular and axenic hypoxic conditions. The inhibition (confirmed using the gold standard colony forming unit assay) was found to increase with "double stimuli" or higher concentration of the extract. Organic solvent extracts were found to inhibit bacterial growth more than the aqueous extracts under microaerophilic conditions mimicked through axenic and intracellular assays. This could be further explored to evaluate the potential of the plant to be used against nonreplicating/dormant bacilli. © The Author(s) 2014.

  5. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Li Luen-Luen

    2011-08-01

    Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  6. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    KAUST Repository

    Sabella, Stefania

    2014-04-09

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

  7. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    KAUST Repository

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-01-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

  8. Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

    Science.gov (United States)

    Xing, Li; McDonald, Joseph J; Kolodziej, Steve A; Kurumbail, Ravi G; Williams, Jennifer M; Warren, Chad J; O'Neal, Janet M; Skepner, Jill E; Roberds, Steven L

    2011-03-10

    Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

  9. Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles in proliferating and nonproliferating mammalian cells

    International Nuclear Information System (INIS)

    Korbelik, M.; Osmak, M.; Suhar, A.; Turk, V.; Skrk, J.

    1990-01-01

    Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles were examined in proliferating and nonproliferating Chinese hamster fibroblasts (V 79). The results show that there are significant alterations in cysteine and aspartic intracellular proteinases activity already in the early postirradiation period, which are different in proliferating and nonproliferating cells. Irradiation of the cells examined to low doses and up to 15 Gy induced an increase in cysteine proteinases activity in the early postexposure period, while at higher irradiation doses applied, the activity of these proteinases was decreased. These observations suggest that intracellular proteinases are actively participating in process involving recovery from radiation injury or cell killing. (orig.) [de

  10. Meta-analysis of microsomal epoxide hydrolase gene polymorphism and risk of hepatocellular carcinoma.

    Science.gov (United States)

    Zhong, Jian-Hong; Xiang, Bang-De; Ma, Liang; You, Xue-Mei; Li, Le-Qun; Xie, Gui-Sheng

    2013-01-01

    Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH). Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent. PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04-1.75, p = 0.02), homozygote comparison (OR = 1.65, 95% CI = 1.07-2.54, p = 0.02) and a recessive genetic model (OR = 1.54, 95% CI = 1.21-1.96, penvironment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.

  11. Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

    Science.gov (United States)

    Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

    2009-01-01

    Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

  12. Single quantum dot tracking reveals the impact of nanoparticle surface on intracellular state.

    Science.gov (United States)

    Zahid, Mohammad U; Ma, Liang; Lim, Sung Jun; Smith, Andrew M

    2018-05-08

    Inefficient delivery of macromolecules and nanoparticles to intracellular targets is a major bottleneck in drug delivery, genetic engineering, and molecular imaging. Here we apply live-cell single-quantum-dot imaging and tracking to analyze and classify nanoparticle states after intracellular delivery. By merging trajectory diffusion parameters with brightness measurements, multidimensional analysis reveals distinct and heterogeneous populations that are indistinguishable using single parameters alone. We derive new quantitative metrics of particle loading, cluster distribution, and vesicular release in single cells, and evaluate intracellular nanoparticles with diverse surfaces following osmotic delivery. Surface properties have a major impact on cell uptake, but little impact on the absolute cytoplasmic numbers. A key outcome is that stable zwitterionic surfaces yield uniform cytosolic behavior, ideal for imaging agents. We anticipate that this combination of quantum dots and single-particle tracking can be widely applied to design and optimize next-generation imaging probes, nanoparticle therapeutics, and biologics.

  13. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    Science.gov (United States)

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited. © 2013 FEBS.

  14. Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

    DEFF Research Database (Denmark)

    Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

    2011-01-01

    The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

  15. Rapid extra-/intracellular biosynthesis of gold nanoparticles by the fungus Penicillium sp.

    Science.gov (United States)

    Du, Liangwei; Xian, Liang; Feng, Jia-Xun

    2011-03-01

    In this work, the fungus Penicillium was used for rapid extra-/intracellular biosynthesis of gold nanoparticles. AuCl4 - ions reacted with the cell filtrate of Penicillium sp. resulting in extracellular biosynthesis of gold nanoparticles within 1 min. Intracellular biosynthesis of gold nanoparticles was obtained by incubating AuCl4 - solution with fungal biomass for 8 h. The gold nanoparticles were characterized by means of visual observation, UV-Vis absorption spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). The extracellular nanoparticles exhibited maximum absorbance at 545 nm in UV-Vis spectroscopy. The XRD spectrum showed Bragg reflections corresponding to the gold nanocrystals. TEM exhibited the formed spherical gold nanoparticles in the size range from 30 to 50 nm with an average size of 45 nm. SEM and TEM revealed that the intracellular gold nanoparticles were well dispersed on the cell wall and within the cell, and they are mostly spherical in shape with an average diameter of 50 nm. The presence of gold was confirmed by EDX analysis.

  16. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  17. Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy

    OpenAIRE

    Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

    2013-01-01

    Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellu...

  18. Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

    Full Text Available ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1 is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

  19. Factors influencing intracellular uptake and radiosensitization by 2-nitroimidazoles in vitro

    International Nuclear Information System (INIS)

    Brown, D.M.; Gonzalez-Mendez, R.; Brown, J.M.

    1983-01-01

    In this study it is shown that the radiosensitization of hypoxic Chinese hamster ovary (HA-1) cells in vitro by misonidazole (MIS) and other 1-substituted 2-nitroimidazoles depends on the rate and extent of intracellular uptake of these radiosensitizers, which in turn is governed by their lipophilicity [expressed as the octanol:water partition coefficient (P)]. As the lipophilicity of the compounds decreased, the rate of drug entry into the cells was slower, and below P values of approximately 0.05, peak intracellular drug concentrations were found to be lower than that of MIS (P=0.43). In addition, the number of hydroxyl groups on the side chain of the nitroimidazole molecule influenced the uptake of drug into the cells. For compounds of similar P, but differing in the number of side-chain hydroxyl groups, the addition of a single hydroxyl group to the molecule decreased the amount of drug entering the cell by a factor of approximately 2. These compounds enter the cell by nonmediated passive diffusion since altering the energy (ATP) capacity of the cell by 2-deoxyglucose did not affect uptake. It is also shown that increases in temperature or decreases in pH can increase the intracellular uptake of MIS. For example, equal intracellular and extracellular concentrations (100% uptake) of MIS were obtained if cells were heated to 44-45 0 C for 15 min compared to 20-40% uptake at 37 0 C. Increases in MIS uptake by factors of 2 to 3 could be demonstrated within 30 min when cells were incubated in Hanks' balanced salt solution at pH between 6.0 and 6.3 without loss of cell viability. In addition, MIS uptake in aerobic cultured cells varied from 15 to 60% depending on the cell line and culure conditions used

  20. Impact of intracellular metallothionein on metal biouptake and partitioning dynamics at bacterial interfaces.

    Science.gov (United States)

    Présent, Romain M; Rotureau, Elise; Billard, Patrick; Pagnout, Christophe; Sohm, Bénédicte; Flayac, Justine; Gley, Renaud; Pinheiro, José P; Duval, Jérôme F L

    2017-11-08

    Genetically engineered microorganisms are alternatives to physicochemical methods for remediation of metal-contaminated aquifers due to their remarkable bioaccumulation capacities. The design of such biosystems would benefit from the elaboration of a sound quantitative connection between performance in terms of metal removal from aqueous solution and dynamics of the multiscale processes leading to metal biouptake. In this work, this elaboration is reported for Escherichia coli cells modified to overexpress intracellular metallothionein (MTc), a strong proteinaceous metal chelator. Depletion kinetics of Cd(ii) from bulk solution following biouptake and intracellular accumulation is addressed as a function of cell volume fraction using electroanalytical probes and ligand exchange-based analyses. It is shown that metal biouptake in the absence and presence of MTc is successfully interpreted on the basis of a formalism recently developed for metal partitioning dynamics at biointerfaces with integration of intracellular metal speciation. The analysis demonstrates how fast sequestration of metals by intracellular MTc bypasses metal excretion (efflux) and enhances the rate of metal depletion to an extent such that complete removal is achieved at sufficiently large cell volume fractions. The magnitude of the stability constant of nanoparticulate metal-MTc complexes, as derived from refined analysis of macroscopic bulk metal depletion data, is further confirmed by independent electrochemical measurement of metal binding by purified MTc extracts.

  1. A new type of intracellular retention signal identified in a pestivirus structural glycoprotein.

    Science.gov (United States)

    Burrack, Sandra; Aberle, Daniel; Bürck, Jochen; Ulrich, Anne S; Meyers, Gregor

    2012-08-01

    Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.

  2. Reduced intracellular c-di-GMP content increases expression of quorum sensing-regulated genes in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Li, Yingying

    2017-01-01

    Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm...... cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents...... possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP...

  3. Intracellular ion monitoring using a gold-core polymer-shell nanosensor architecture

    Energy Technology Data Exchange (ETDEWEB)

    Stanca, S E; Cranfield, C G; Biskup, C [Biomolecular Photonics Group, University Hospital Jena, Teichgraben 8, 07743 Jena (Germany); Nietzsche, S [Centre for Electron Microscopy, University Hospital Jena, Ziegel-muehlenweg 1, 07743 Jena (Germany); Fritzsche, W, E-mail: sarmiza.stanca@mti.uni-jena.de, E-mail: charles.cranfield@mti.uni-jena.de, E-mail: christoph.biskup@mti.uni-jena.de [Institute of Photonic Technology, Albert-Einstein-Strasse 9, 07745 Jena (Germany)

    2010-02-05

    In this study, we describe the design of new ratiometric fluorescent nanosensors, whose architecture is based on a gold core surrounded by a poly(vinyl alcohol)-polyacetal shell. To the gold core, indicator dyes and reference dyes are attached via a cysteine linker. This nanosensor architecture is flexible with regards to the number and types of fluorophore linkages possible. The robust poly(vinyl alcohol)-polyacetal shell protects the fluorophores linked to the core from non-specific interactions with intracellular proteins. The nanosensors developed in this way are biocompatible and can be easily incorporated into mammalian cells without the use of transfection agents. Here, we show the application of these nanosensors for intracellular pH and sodium ion measurements.

  4. Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death

    Directory of Open Access Journals (Sweden)

    Zheng-Wei Lee

    2017-10-01

    Full Text Available Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4, or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

  5. Inhibition of SAH-hydrolase during tobacco seeds germination induced by treatment by DHPA leads to mitotically heritable DNA hypomethylation, ectopic expression of floral genes and floral whorl malformations

    Czech Academy of Sciences Publication Activity Database

    Fulneček, Jaroslav; Matyášek, Roman; Kabáthová, E.; Votruba, Ivan; Holý, Antonín; Kovařík, Aleš

    2013-01-01

    Roč. 280, Suppl. 1 (2013), s. 522-522 ISSN 1742-464X. [Congress of the Federation of European Biochemical Societies (FEBS) /38./. 06.07.2013-11.07.2013, Saint Petersburg] R&D Projects: GA ČR GBP501/12/G090; GA ČR GA206/09/1751; GA ČR GA13-10057S Institutional support: RVO:68081707 ; RVO:61388963 Keywords : SAH-hydrolase * DNA hypomethylation * DHPA Subject RIV: CE - Biochemistry

  6. Development of an in vitro photosafety evaluation method utilizing intracellular ROS production in THP-1 cells.

    Science.gov (United States)

    Toyoda, Akemi; Itagaki, Hiroshi

    2018-01-01

    Photoreactive compounds that may experience exposure to ultraviolet (UV) radiation can lead to the intracellular production of reactive oxygen species (ROS), which may cause phototoxic and photoallergenic responses. Here, we developed a novel in vitro photosafety assay and investigated whether it could be used to predict phototoxicity and photosensitivity by measuring changes in intracellular ROS production. THP-1 cells that had previously taken up 5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H 2 DFFDA), a ROS-sensitive fluorescent reagent, were exposed to photoreactive substances such as phototoxic and photoallergenic materials and then subjected to with UV-A irradiation (5 J/cm 2 ). The fluorescence intensity was subsequently measured using a flow cytometer, and the intracellular ROS production was calculated. A statistically significant increase in ROS following treatment with photoreactive substances was observed in cells irradiated with UV-A. In contrast, no significant increase was observed for non-photoreactive substances in comparison to the control solution. Next, to confirm the impact of intracellular ROS on the photosensitive response, changes in CD86 and CD54 expression were measured following quencher addition during the photo human cell line activation test (photo h-CLAT). The results confirmed the reduction of CD86 and CD54 expression in response to photoallergenic substances following quencher addition. Together, these findings suggest that intracellular ROS production is involved in photosensitizing reactions. Therefore, we suggest that the developed method utilizing intracellular ROS production as an index may be useful as a novel in vitro evaluation tool for photoreactive substances.

  7. Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules

    DEFF Research Database (Denmark)

    Troelsen, J T; Mitchelmore, C; Sjöström, H

    1994-01-01

    Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are enterocyte-specific gene products. The identification of regulatory cis-elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal-specific nuclear factors (NF-LPH1...... and SIF1-BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis-elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 k......Da oligomeric complexes. Based on these results NF-LPH1 and SIF1-BP are suggested to be either identical or closely related molecules....

  8. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  9. Monitoring Intracellular Redox Changes in Ozone-exposed airway epithelial cells

    Science.gov (United States)

    Background: The toxicity of many compounds involves oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically-encoded probes designed for monitoring intracellular redox s...

  10. A Bombesin-Shepherdin Radioconjugate Designed for Combined Extra- and Intracellular Targeting

    Directory of Open Access Journals (Sweden)

    Christiane A. Fischer

    2014-05-01

    Full Text Available Radiolabeled peptides which target tumor-specific membrane structures of cancer cells represent a promising class of targeted radiopharmaceuticals for the diagnosis and therapy of cancer. A potential drawback of a number of reported radiopeptides is the rapid washout of a substantial fraction of the initially delivered radioactivity from cancer cells and tumors. This renders the initial targeting effort in part futile and results in a lower imaging quality and efficacy of the radiotracer than achievable. We are investigating the combination of internalizing radiopeptides with molecular entities specific for an intracellular target. By enabling intracellular interactions of the radioconjugate, we aim at reducing/decelerating the externalization of radioactivity from cancer cells. Using the “click-to-chelate” approach, the 99mTc-tricarbonyl core as a reporter probe for single-photon emission computed tomography (SPECT was combined with the binding sequence of bombesin for extracellular targeting of the gastrin-releasing peptide receptor (GRP-r and peptidic inhibitors of the cytosolic heat shock 90 protein (Hsp90 for intracellular targeting. Receptor-specific uptake of the multifunctional radioconjugate could be confirmed, however, the cellular washout of radioactivity was not improved. We assume that either endosomal trapping or lysosomal degradation of the radioconjugate is accountable for these observations.

  11. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  12. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    Science.gov (United States)

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  13. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    International Nuclear Information System (INIS)

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-01-01

    Highlights: ► We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. ► 3-D images of TAT-SPIONs in a cell are clearly shown. ► Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  14. Metabolism of Cartilage Proteoglycans in Health and Disease

    Directory of Open Access Journals (Sweden)

    Demitrios H. Vynios

    2014-01-01

    Full Text Available Cartilage proteoglycans are extracellular macromolecules with complex structure, composed of a core protein onto which a variable number of glycosaminoglycan chains are attached. Their biosynthesis at the glycosaminoglycan level involves a great number of sugar transferases well-orchestrated in Golgi apparatus. Similarly, their degradation, either extracellular or intracellular in lysosomes, involves a large number of hydrolases. A deficiency or malfunction of any of the enzymes participating in cartilage proteoglycan metabolism may lead to severe disease state. This review summarizes the findings regarding this topic.

  15. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  16. Horizontal Transmission of Intracellular Insect Symbionts via Plants

    Directory of Open Access Journals (Sweden)

    Ewa Chrostek

    2017-11-01

    Full Text Available Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  17. Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

    Directory of Open Access Journals (Sweden)

    Jiang LQ

    2017-08-01

    Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

  18. Fully glutathione degradable waterborne polyurethane nanocarriers: Preparation, redox-sensitivity, and triggered intracellular drug release

    Energy Technology Data Exchange (ETDEWEB)

    Omrani, Ismail; Babanejad, Niloofar; Shendi, Hasan Kashef; Nabid, Mohammad Reza, E-mail: m-nabid@sbu.ac.ir

    2017-01-01

    Polyurethanes are important class of biomaterials that are extensively used in medical devices. In spite of their easy synthesis, polyurethanes that are fully degradable in response to the intracellular reducing environment are less explored for controlled drug delivery. Herein, a novel glutathione degradable waterborne polyurethane (WPU) nanocarrier for redox triggered intracellular delivery of a model lipophilic anticancer drug, doxorubicin (DOX) is reported. The WPU was prepared from polyaddition reaction of isophorone diisocyanate (IPDI) and a novel linear polyester polyol involving disulfide linkage, disulfide labeled chain extender, dimethylolpropionic acid (DMPA) using dibutyltin dilaurate (DBTDL) as a catalyst. The resulting polyurethane self-assembles into nanocarrier in water. The dynamic light scattering (DLS) measurements and scanning electron microscope (SEM) revealed fast swelling and disruption of nanocarriers under an intracellular reduction-mimicking environment. The in vitro release studies showed that DOX was released in a controlled and redox-dependent manner. MTT assays showed that DOX-loaded WPU had a high in vitro antitumor activity in both HDF noncancer cells and MCF- 7 cancer cells. In addition, it is found that the blank WPU nanocarriers are nontoxic to HDF and MCF-7 cells even at a high concentration of 2 mg/mL. Hence, nanocarriers based on disulfide labeled WPU have appeared as a new class of biocompatible and redox-degradable nanovehicle for efficient intracellular drug delivery. - Highlights: • A novel fully glutathione degradable waterborne polyurethane was developed. • The waterborne nanocarrier with disulfide bonds in both hard and soft segment were developed for redox-triggered intracellular delivery of DOX. • The polyester diol bearing disulfide bonds in the backbone was prepared by a polycondensation polymerization reaction.

  19. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  20. LDL Receptors as Gateways for Intracellular Porphyrin Uptake

    International Nuclear Information System (INIS)

    Novick, S.; Laster, B.; Quastel, M.

    2004-01-01

    Boronated compounds are currently being studied for possible use in Boron Neutron Capture Therapy (BNCT). We found that one of these agents, BOPP (tetrakis-carborane-carboxylate, esters of 2,4-bis (a,b- dihydroxyethyl) deuteroporphyrin IX), could also be labeled with indium (In-BOPP) and, therefore, could also be used potentially to transport high Z atoms into tumor cell DNA for AET (Auger Electron Therapy). In order to assess the uptake of these agents into cells, the role of the LDL receptor in the intracellular accumulation of BOPP and In-BOPP was investigated. Pre-incubation of V-79 Chinese hamster cells in medium containing delipidized fetal bovine serum (FBS) markedly increased the subsequent uptake of intracellular boron transported by both BOPP and In-BOPP when compared with cells that had been pre-incubated with medium containing 10% normal FBS (lipidized). The increased uptake was characterized by elevated levels of receptor, and greater affinity was shown for both BOPP and In-BOPP, although less marked with the latter. Positive cooperativity was demonstrated by sigmoid saturation curves, Scatchard analysis and Hill plots. Increasing the amount of LDL in the incubation medium had a relatively small effect on the total accumulation of either indium or boron atoms inside the cell. Furthermore, chemical acetylation of LDL did not decrease the intracellular uptake of either boron or indium transported by BOPP or In-BOPP. It is thus concluded that BOPP and In-BOPP preferentially enter the cells directly by way of the LDL receptor and that only a small fraction of these molecules are transported into the cells indirectly using serum LDLs as their carriers. These data suggest a novel way of bringing greater amounts of boron and indium (and perhaps other agents) into tissues. Porphyrins can be used to transport different agents into tumor cells because they are tumor affinic molecules. Tumors express a higher number of LDL receptors than do most normal tissues