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Sample records for extracellular histidine residues

  1. Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity.

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    Shotaro Sasaki

    Full Text Available Monocarboxylate transporter 4 (MCT4 is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity.

  2. The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

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    Husain, S. S.; Lowe, G.

    1970-01-01

    Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

  3. Agglutination of human erythrocytes by the interaction of Zn(2+)ion with histidine-651 on the extracellular domain of band 3.

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    Kiyotake, Kento; Ochiai, Hideharu; Yamaguchi, Takeo

    2016-05-01

    Clustering of band 3, chloride/bicarbonate exchanger, has been reported in Zn(2+)-treated human erythrocytes. However, the agglutination of human erythrocytes is also induced by the interaction of Zn(2+)ion with histidine on band 3. Identification of histidine that interacts with Zn(2+)ion remains to be determined. The Zn(2+)-induced agglutination of human erythrocytes was unaffected by chymotrypsin cleavage of the small loop region containing His-547 in the extracellular domain of band 3. On the other hand, papain digestion of the large loop region containing His-651 in band 3 inhibited such Zn(2+)-induced agglutination. Moreover, Zn(2+)-induced erythrocyte agglutination was inhibited by the peptide (ARGWVIHPLG) containing His-651, but not by the peptide such as ARGWVIRPLG, which His-651 was substituted by arginine. Among 10 kinds of animal erythrocytes tested, interestingly, no agglutination by Zn(2+)ions was observed in cow cells only that the forth amino acid in the upstream from His-669 on the large loop of cow band 3 is aspartate (Asp-665) instead of glycine. As expected, the agglutination of human erythrocytes by Zn(2+) ions was inhibited in the presence of aspartate. These data indicate that the interaction of Zn(2+) ion with His-651 residue of band 3 plays an important role in the Zn(2+)-induced agglutination of human erythrocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

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    Buku, Angeliki; Mendlowitz, Milton; Condie, Barry A; Price, Joseph A

    2004-06-01

    The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.

  5. pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

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    Yeo, Kwon Joo [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Kim, Eun Hye [Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-Ro, Yuseong-Gu, Daejeon 305-333 (Korea, Republic of); Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Kwon, Ohsuk [Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-Ro, Yuseong-Gu, Daejeon 305-333 (Korea, Republic of); Hong, Young-Soo [Chemical Biology Research Center, KRIBB, 30 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Cheong, Chaejoon, E-mail: cheong@kbsi.re.kr [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Cheong, Hae-Kap, E-mail: haekap@kbsi.re.kr [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of)

    2013-02-15

    Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.

  6. Production of extracellular amylase from agricultural residues by a ...

    African Journals Online (AJOL)

    The production of extracellular amylases by solid state fermentation (SSF) was investigated employing our laboratory isolate Aspergillus sp.MK07. Various agricultural residual substrates like wheat bran, rice bran and green gram husk were studied for enzyme production. Highest enzyme production was obtained with ...

  7. Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

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    Parker, Antony R

    2003-10-01

    The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

  8. Imidazole Nitrogens of Two Histidine Residues Participating in N-H···N Hydrogen Bonds in Protein Structures: Structural Bioinformatics Approach Combined with Quantum Chemical Calculations.

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    Iyer, Abhishek Hariharan; Krishna Deepak, R N V; Sankararamakrishnan, Ramasubbu

    2018-01-25

    Protein structures are stabilized by different types of hydrogen bonds. However, unlike the DNA double helical structure, the N-H···N type of hydrogen bonds is relatively rare in proteins. N-H···N hydrogen bonds formed by imidazole groups of two histidine residues have not been investigated. We have systematically analyzed 5333 high-resolution protein structures with resolution 1.8 Å or better and identified 285 histidine pairs in which the nitrogen atoms of the imidazole side chains can potentially participate in N-H···N hydrogen bonds. The histidine pairs were further divided into two groups, neutral-neutral and protonated-neutral, depending on the protonation state of the donor histidine. Quantum chemical calculations were performed on imidazole groups adopting the same geometry observed in the protein structures. Average interaction energies between the interacting imidazole groups are -6.45 and -22.5 kcal/mol for neutral-neutral and protonated-neutral, respectively. Hydrogen bond interaction between the imidazole moieties is further confirmed by natural bond orbital analyses of the model compounds. Histidine residues involved in N-H···N hydrogen bonds are relatively more buried and have low B-factor values in the protein structures. N-H···N hydrogen bond formed by a pair of buried histidine residues can significantly contribute to the structural stability of proteins.

  9. Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

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    Sabina Eigenbrod

    Full Text Available Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC at octapeptide repeat (OR and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele or at site 5 (composed of residues His-95 and His-110; "H95G" allele and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G at levels comparable to wild-type (wt controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G mice and diffuse PrPSc deposition in (TgPrP(H95G mice, were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

  10. Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein▿

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    Qin, Zhao-ling; Zheng, Yan; Kielian, Margaret

    2009-01-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion. PMID:19244325

  11. Role of conserved histidine residues in the low-pH dependence of the Semliki Forest virus fusion protein.

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    Qin, Zhao-Ling; Zheng, Yan; Kielian, Margaret

    2009-05-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.

  12. Chiral recognition of proteins having L-histidine residues on the surface with lanthanide ion complex incorporated-molecularly imprinted fluorescent nanoparticles

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    Uzun, Lokman, E-mail: lokman@hacettepe.edu.tr [Hacettepe University, Department of Chemistry, 06381, Ankara (Turkey); Uzek, Recep; Şenel, Serap [Hacettepe University, Department of Chemistry, 06381, Ankara (Turkey); Say, Ridvan [Anadolu University, Department of Chemistry, 26470, Eskisehir (Turkey); Denizli, Adil [Hacettepe University, Department of Chemistry, 06381, Ankara (Turkey)

    2013-08-01

    In this study, lanthanide ion complex incorporated molecularly imprinted fluorescent nanoparticles were synthesized. A combination of three novel approaches was applied for the purpose. First, lanthanide ions [Terbium(III)] were complexed with N-methacryloyl-L-histidine (MAH), polymerizable derivative of L-histidine amino acid, in order to incorporate the complex directly into the polymeric backbone. At the second stage, L-histidine molecules imprinted nanoparticles were utilized instead of whole protein imprinting in order to avoid whole drawbacks such as fragility, complexity, denaturation tendency, and conformation dependency. At the third stage following the first two steps mentioned above, imprinted L-histidine was coordinated with cupric ions [Cu(II)] to conduct the study under mild conditions. Then, molecularly imprinted fluorescent nanoparticles synthesized were used for L-histidine adsorption from aqueous solution to optimize conditions for adsorption and fluorimetric detection. Finally, usability of nanoparticles was investigated for chiral biorecognition using stereoisomer, D-histidine, racemic mixture, D,L-histidine, proteins with surface L-histidine residue, lysozyme, cytochrome C, or without ribonuclease A. The results revealed that the proposed polymerization strategy could make significant contribution to the solution of chronic problems of fluorescent component introduction into polymers. Additionally, the fluorescent nanoparticles reported here could be used for selective separation and fluorescent monitoring purposes. Highlights: • Lanthanide ion complex incorporated molecularly imprinted fluorescent nanoparticles • Direct incorporation of the fluorescent complex into polymeric backbone. • Imprinting by assistance of cupric ion coordination into nanoparticles • Evaluation of the chiral biorecognition ability of nanoparticles • Simultaneous selective separation and fluorescent monitoring.

  13. The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.

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    Arjomand, Maryam Rezaei; Ahmadian, Gholamreza; Habibi-Rezaei, Mehran; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Moosavi-Movahedi, Ali Akbar; Amanlou, Massoud

    2017-05-01

    Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure ( 189 AELH 192 ) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)*

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    Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

    2012-01-01

    Zinc activates a specific Zn2+-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca2+ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na+/H+ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn2+ binding site, His17 or His19, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp313 with alanine resulted in similar Ca2+ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na+/H+ exchange at pH 7.4 and pH 6.5. Substitution of Asp313 to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp313, which was shown to modulate Zn2+ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity. PMID:22879599

  15. The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

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    Helene J Bustad

    Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

  16. Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function, Modulating pH Sensitivity, Transporter Kinetics, Na+ Cooperativity, Conformational Stability, and Subcellular Localization*

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    Ormazabal, Valeska; Zuñiga, Felipe A.; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M.; Vera, Juan Carlos; Rivas, Coralia I.

    2010-01-01

    Na+-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His109, His203, His206, His269, and His413, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His413, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na+ and loss of Na+ cooperativity, which leads to a decreased Vmax without altering the transport Km; (ii) exofacial histidine residues His203, His206, and His413 may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport Km; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function. PMID:20843809

  17. Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

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    Kwon Joo Yeo

    Full Text Available The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

  18. Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

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    Yeo, Kwon Joo; Hong, Young-Soo; Jee, Jun-Goo; Lee, Jae Kyoung; Kim, Hyo Jeong; Park, Jin-Wan; Kim, Eun-Hee; Hwang, Eunha; Kim, Sang-Yoon; Lee, Eun-Gyeong; Kwon, Ohsuk; Cheong, Hae-Kap

    2014-01-01

    The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

  19. Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase.

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    Anderson, P M; Korte, J J; Holcomb, T A; Cho, Y G; Son, C M; Sung, Y C

    1994-05-27

    Reaction of the single cysteine residue in each subunit of cyanase with certain SH reagents gives an active decameric derivative that dissociates reversibly to an inactive dimer derivative (Anderson, P. M., Johnson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1988) J. Biol. Chem. 263, 5674-5680). Reaction of mixed disulfide dimer derivatives of cyanase with dithiothreitol at 0 degree C results in formation of a disulfide bond between the subunits in the dimer. The disulfide dimer was inactive and did not associate to a decamer; the intersubunit disulfide bond could not be formed when the dimers were associated as a decamer. The two SH groups apparently are in close proximity to each other in the dissociated dimer but not when the dimer is associated to a decamer. Substitution of glycine for the cysteine residue or of tyrosine, asparagine, glycine, valine, or leucine for the single histidine residue in each subunit gave mutant enzymes that were active. However, H113N, H113Y, and C83G were unstable at low temperature and/or ionic strength, dissociating reversibly to an inactive dimer. Efficient reassociation required the presence of bicarbonate or cyanate analog. The results are consistent with a proposed single site per subunit model explaining apparent half-site binding of substrates and the requirement of decameric structure for activity.

  20. pKa determination of histidine residues in α-conotoxin MII peptides by 1H NMR and constant pH molecular dynamics simulation.

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    McDougal, Owen M; Granum, David M; Swartz, Mark; Rohleder, Conrad; Maupin, C Mark

    2013-03-07

    α-Conotoxin MII (α-CTxMII) is a potent and selective peptide antagonist of neuronal nicotinic acetylcholine receptors (nAChR's). Studies have shown that His9 and His12 are significant determinants of toxin binding affinity for nAChR, while Glu11 may dictate differential toxin affinity between nAChR isoforms. The protonation state of these histidine residues and therefore the charge on the α-CTx may contribute to the observed differences in binding affinity and selectivity. In this study, we assess the pH dependence of the protonation state of His9 and His12 by (1)H NMR spectroscopy and constant pH molecular dynamics (CpHMD) in α-CTxMII, α-CTxMII[E11A], and the triple mutant, α-CTxMII[N5R:E11A:H12K]. The E11A mutation does not significantly perturb the pKa of His9 or His12, while N5R:E11A:H12K results in a significant decrease in the pKa value of His9. The pKa values predicted by CpHMD simulations are in good agreement with (1)H NMR spectroscopy, with a mean absolute deviation from experiment of 0.3 pKa units. These results support the use of CpHMD as an efficient and inexpensive predictive tool to determine pKa values and structural features of small peptides critical to their function.

  1. Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread

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    He Yong

    2012-09-01

    Full Text Available Abstract Background A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV. Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus. Results By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture. Conclusions These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may

  2. Contribution of individual histidines to the global stability of human prolactin.

    Science.gov (United States)

    Keeler, Camille; Tettamanzi, M Cristina; Meshack, Syrus; Hodsdon, Michael E

    2009-05-01

    A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH-dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH-dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2-3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.

  3. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues.

    Science.gov (United States)

    Britsch, L; Dedio, J; Saedler, H; Forkmann, G

    1993-10-15

    A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

  4. An extracellular fungal polysaccharide composed of 2-acetamido-2-deoxy-D-glucuronic acid residues.

    Science.gov (United States)

    Watson, P R; Sandford, P A; Burton, K A; Cadmus, M C; Jeanes, A

    1976-02-01

    The black yeast-like fungus NRRL YB-4163, now tentatively identified as Rhinocladiella elatior Mangenot, has been found to produce an extracellular microbial polysaccharide composed mainly of 2-acetamido-2-deoxy-D-glucuronic acid residues. Polysaccharide (PS) YB-4163, when isolated in good yield as the neutral potassium salt, dissolves readily in water to produce extremely viscous solutions, which form stable foams and emulsions. By depolymerizing PS YB-4163 with [14C]methanol-HCl, the polysaccharide can be both identified and quantitated radiochemically by determining the individual [14C]methyl glycosides after their separation by paper chromatography. When the methyl glycosides of PS YB-4163 were reduced with NaB3H4, only the methyl glycosides of 2-acetamido-2-deoxy-D-[6-3H]glucose were found. Analysis of the monosaccharide released from carboxyl-reduced PS YB-4163 by acid hydrolysis or methanolysis also showed 2-acetamido-2-deoxy-D-glucuronic acid to be the main constituent. Previously, the only polysaccharides known to be composed entirely or hexosaminuronic acid have been cellular products from pathogens. Of these, the antigenic polysaccharide (SPSA) from Staphylococcus aureus is composed entirely of 2-amino-2-deoxy-D-glucuronic acid, but its amino groups are substituted equally with acetyl and N-acetylalanyl groups. The specific optical rotation of PS YB-4163, [alpha]20D -75 degrees (c 0.5, water), is similar to that of SPSA (-91 degrees), and suggests beta-D-linkages that must be either (1 leads to 3) or (1 leads to 4).

  5. Identification of active-site residues in Aspergillus ficuum extracellular pH 2.5 optimum acid phosphatase.

    Science.gov (United States)

    Ullah, A H; Dischinger, H C

    1993-04-30

    Primary structure elucidation of peptides generated by cyanogen bromide, endoproteinase Glu-C, and clostripain cleavage of an Aspergillus ficuum extracellular pH optimum 2.5 acid phosphatase identified a region which contains the active site of the enzyme. The 23-residue segment contains the fragment RHGXRXP, which is homologous to acid phosphatase from Saccharomyces spp., Aspergillus ficuum, mammals, and bacteria. Homologous or conservative substitutions are observed in the 10-amino acid fragment preceding this region.

  6. Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface

    National Research Council Canada - National Science Library

    Ennion, Steven J; Evans, Richard J

    2002-01-01

    P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor...

  7. Conserved Cysteine Residues in the Extracellular Loop of the Human P2X1 Receptor Form Disulfide Bonds and Are Involved in Receptor Trafficking to the Cell Surface

    National Research Council Canada - National Science Library

    Steven J. Ennion; Richard J. Evans

    2002-01-01

    P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X 1 receptor...

  8. Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

    Directory of Open Access Journals (Sweden)

    Milos B Rokic

    Full Text Available The binding of ATP to trimeric P2X receptors (P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

  9. UTILIZATION OF AN ACTIVE SERINE 101 -] CYSTEINE MUTANT TO DEMONSTRATE THE PROXIMITY OF THE CATALYTIC SERINE 101 AND HISTIDINE 237 RESIDUES IN THIOESTERASE-II

    NARCIS (Netherlands)

    WITKOWSKI, A; NAGGERT, J; WITKOWSKA, HE; RANDHAWA, ZI; SMITH, S

    1992-01-01

    Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue

  10. Carboplatin binding to histidine

    NARCIS (Netherlands)

    Tanley, Simon W M; Diederichs, Kay; Kroon - Batenburg, Louise|info:eu-repo/dai/nl/070944172; Levy, Colin; Schreurs, Antoine M M|info:eu-repo/dai/nl/304847453; Helliwell, John R.

    2014-01-01

    Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl

  11. The dapE-encoded N-succinyl-L,L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

    Science.gov (United States)

    Gillner, Danuta M.; Bienvenue, David L.; Nocek, Boguslaw P.; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C.

    2009-01-01

    The catalytic and structural properties of the H67A and H349A altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from H. influenzae were investigated. Based on sequence alignment with CPG2 both H67 and H349 were predicted to be Zn(II) ligands. Catalytic activity was observed for the H67A altered DapE enzyme which exhibited kcat = 1.5 ± 0.5 sec−1 and Km = 1.4 ± 0.3 mM. No catalytic activity was observed for H349A under the experimental conditions used. The EPR and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to WT DapE after the addition of a single Co(II) ion. The addition of one equivalent of Co(II) to H67A altered DapE provides spectra that are very different from the first Co(II) binding site of the WT enzyme, but similar to the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from N. meningitidis as a template and superimposed on the structure of AAP. This homology structure confirms the assignment of H67 and H349 as active site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP, all of the amino acid residues of DapE are nearly identical. PMID:18712420

  12. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.

    Science.gov (United States)

    Gillner, Danuta M; Bienvenue, David L; Nocek, Boguslaw P; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C

    2009-01-01

    The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

  13. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  14. Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

    Science.gov (United States)

    Elliott, Candace; Müller, Judith; Miklis, Marco; Bhat, Riyaz A; Schulze-Lefert, Paul; Panstruga, Ralph

    2005-01-01

    We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

  15. Conserved extracellular cysteine residues and cytoplasmic loop–loop interplay are required for functionality of the heptahelical MLO protein

    Science.gov (United States)

    Elliott, Candace; Müller, Judith; Miklis, Marco; Bhat, Riyaz A.; Schulze-Lefert, Paul; Panstruga, Ralph

    2004-01-01

    We performed a structure–function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop–loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family. PMID:15352871

  16. Carboplatin binding to histidine

    Science.gov (United States)

    Tanley, Simon W. M.; Diederichs, Kay; Kroon-Batenburg, Loes M. J.; Levy, Colin; Schreurs, Antoine M. M.; Helliwell, John R.

    2014-01-01

    Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described. PMID:25195881

  17. Formation of RNA phosphodiester bond by histidine-containing dipeptides

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Dörr, Mark; Chotera, Agata

    2013-01-01

    A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L-dipeptides containing a histidine residue, primarily Ser-His, were used. Reactions were carried out ...

  18. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

    DEFF Research Database (Denmark)

    Barington, Line; Rummel, Pia C; Lückmann, Michael

    2016-01-01

    and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...... for chemokines. Furthermore, we find that two distinct aromatic residues in ECL2, Y184 (Cys+1) and Y187 (Cys+4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster...

  19. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  20. Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface.

    Science.gov (United States)

    Ennion, Steven J; Evans, Richard J

    2002-02-01

    P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2- to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 +/- 2.0 and 95.0 +/- 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MTSEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEA-biotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X(1) receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.

  1. Histidine side-chain dynamics and protonation monitored by C-13 CPMG NMR relaxation dispersion

    DEFF Research Database (Denmark)

    Hass, M. A. S.; Yilmaz, A.; Christensen, Hans Erik Mølager

    2009-01-01

    the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from N-15 backbone relaxation measurements. Compared to measurements of backbone nuclei, C-13(epsilon 1) dispersion provides a more direct method to monitor interchanging protonation states...... or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the C-13(epsilon 1) dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains......The use of C-13 NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically C-13 labeled histidine residues in plastocyanin (PCu) from...

  2. Analysis of conformational changes in rhodopsin by histidine hydrogen-deuterium exchange.

    Science.gov (United States)

    Lodowski, David T; Miyagi, Masaru

    2015-01-01

    Hydrogen-deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histidines. This measurement, when accompanied by pH titration, provides both pK as and half-lives (t 1/2) of the HDX reaction for individual histidine residues in proteins. The pK a and t 1/2 values indicate the electrostatic environment and the degree of side-chain solvent accessibility of the histidine residues, respectively. Herein we describe an experimental protocol to characterize rhodopsin by His-HDX-MS. This technique can be used to monitor different states of rhodopsin and might be useful for monitoring longtime scale events in other GPCRs.

  3. Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

    Science.gov (United States)

    Guan, Xiaolu; Zhang, Yao; Yu, Mengmeng; Ren, Chaoqi; Gao, Yanni; Yun, Bingling; Liu, Yongzhen; Wang, Yongqiang; Qi, Xiaole; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Gao, Li; Li, Kai; Pan, Qing; Zhang, Baoshan; Wang, Xiaomei; Gao, Yulong

    2018-01-01

    Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in

  4. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  5. Evidence for histidine in the active sites of ficin and stem-bromelain

    Science.gov (United States)

    Husain, S. S.; Lowe, G.

    1968-01-01

    1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5Å locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only. PMID:5722692

  6. Molecular recognition of histidine tRNA by histidyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Nagatoyo, Yukari; Iwaki, Jun; Suzuki, Satoko; Kuno, Atsushi; Hasegawa, Tsunemi

    2005-01-01

    To investigate the recognition sites of histidine tRNA for histidyl-tRNA synthetase from an extreme hyperthermophilic archaeon, Aeropyrum pernix K1, we examined histidylation activities by using overexpressed histidyl-tRNA synthetase and various histidine tRNA transcripts that were prepared by in vitro transcription system. Results indicated that anticodon was not recognized by the histidyl-tRNA synthetase similar to that of Escherichia coli histidine tRNA recognition system. Discriminator base C73 was weekly recognized and an additional G residue was specifically recognized by the enzyme.

  7. Intra- and interprotein phosphorylation between two-hybrid histidine kinases controls Myxococcus xanthus developmental progression.

    Science.gov (United States)

    Schramm, Andreas; Lee, Bongsoo; Higgs, Penelope I

    2012-07-20

    Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple "two-component" systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program.

  8. Intra- and Interprotein Phosphorylation between Two-hybrid Histidine Kinases Controls Myxococcus xanthus Developmental Progression*

    Science.gov (United States)

    Schramm, Andreas; Lee, Bongsoo; Higgs, Penelope I.

    2012-01-01

    Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple “two-component” systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program. PMID:22661709

  9. Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

    Science.gov (United States)

    Dago, Angel E; Schug, Alexander; Procaccini, Andrea; Hoch, James A; Weigt, Martin; Szurmant, Hendrik

    2012-06-26

    Signal transduction proteins such as bacterial sensor histidine kinases, designed to transition between multiple conformations, are often ruled by unstable transient interactions making structural characterization of all functional states difficult. This study explored the inactive and signal-activated conformational states of the two catalytic domains of sensor histidine kinases, HisKA and HATPase. Direct coupling analyses, a global statistical inference approach, was applied to >13,000 such domains from protein databases to identify residue contacts between the two domains. These contacts guided structural assembly of the domains using MAGMA, an advanced molecular dynamics docking method. The active conformation structure generated by MAGMA simultaneously accommodated the sequence derived residue contacts and the ATP-catalytic histidine contact. The validity of this structure was confirmed biologically by mutation of contact positions in the Bacillus subtilis sensor histidine kinase KinA and by restoration of activity in an inactive KinA(HisKA):KinD(HATPase) hybrid protein. These data indicate that signals binding to sensor domains activate sensor histidine kinases by causing localized strain and unwinding at the end of the C-terminal helix of the HisKA domain. This destabilizes the contact positions of the inactive conformation of the two domains, identified by previous crystal structure analyses and by the sequence analysis described here, inducing the formation of the active conformation. This study reveals that structures of unstable transient complexes of interacting proteins and of protein domains are accessible by applying this combination of cross-validating technologies.

  10. Histidine adsorption on nanostructured cerium oxide

    Energy Technology Data Exchange (ETDEWEB)

    Bercha, Sofiia [Charles University in Prague, Faculty of Mathematics and Physics, Department of Surface and Plasma Science, V Holešovičkách 2, CZ-18000 Prague 8 (Czech Republic); Mali, Gregor [National Institute of Chemistry, Laboratory for Inorganic Chemistry and Technology, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Khalakhan, Ivan; Skála, Tomáš [Charles University in Prague, Faculty of Mathematics and Physics, Department of Surface and Plasma Science, V Holešovičkách 2, CZ-18000 Prague 8 (Czech Republic); Prince, Kevin C. [Elettra-Sincrotrone Trieste S.C.p.A., in Area Science Park, Strada Statale 14, km 163.5, Basovizza, Trieste I-34149 (Italy); Matolín, Vladimír [Charles University in Prague, Faculty of Mathematics and Physics, Department of Surface and Plasma Science, V Holešovičkách 2, CZ-18000 Prague 8 (Czech Republic); Tsud, Nataliya, E-mail: Nataliya.Tsud@mff.cuni.cz [Charles University in Prague, Faculty of Mathematics and Physics, Department of Surface and Plasma Science, V Holešovičkách 2, CZ-18000 Prague 8 (Czech Republic)

    2016-10-15

    Highlights: • The surface of nanostructured ceria was functionalized by histidine. • The molecules were deposited from aqueous solution. • Polycrystalline films and nanoparticles of ceria were used as substrates. • Histidine chemisorbs on the surface via deprotonated carboxylate group. - Abstract: Histidine adsorption from neutral aqueous solution on cerium oxide substrates was studied by photoemission with use of synchrotron radiation, soft X-ray absorption spectroscopy and nuclear magnetic resonance. Polycrystalline oxide films and oxide nanoparticles were used as ceria substrates. Independent of the morphology of the support, histidine binds to the oxide through the carboxylic group while the imidazole ring does not participate in the interface formation. Compared to deposition of molecules by evaporation in vacuum, the presence of the solution during adsorption does not alter the histidine bonding to cerium oxide. The present results clearly demonstrate the applicability of the model (in-situ) studies of the histidine/CeO{sub 2} interface to the biocompatible techniques of cerium oxide functionalization.

  11. Neighbor-directed histidine N(τ) alkylation. A route to imidazolium-containing phosphopeptide macrocycles

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Wen-Jian [National Cancer Inst., Frederick, MD (United States); Park, Jung-Eun [National Cancer Inst., Bethesda, MD (United States); Grant, Robert [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lai, Christopher C. [National Cancer Inst., Frederick, MD (United States); Kelley, James A. [National Cancer Inst., Frederick, MD (United States); Yaffe, Michael B. [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lee, Kyung S. [National Cancer Inst., Bethesda, MD (United States); Burke, Terrence R. [National Cancer Inst., Frederick, MD (United States)

    2015-07-07

    Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. These cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Furthermore, neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

  12. Manganese and cobalt binding in a multi-histidinic fragment.

    Science.gov (United States)

    Peana, Massimiliano; Medici, Serenella; Nurchi, Valeria Marina; Crisponi, Guido; Lachowicz, Joanna Izabela; Zoroddu, Maria Antonietta

    2013-12-14

    The binding of Mn(II) and Co(II) ions to a multi-histidinic peptide, the three repeats (T1R2S3R4S5H6T7S8E9G10)3 portion of Cap43 protein, has been studied. Potentiometric measurements have been used to investigate the protonation equilibria and stoichiometry of the species obtained in a wide range of pH and at a 1 : 1 ligand-to-metal molar ratio. NMR, UV-visible and EPR spectroscopy techniques have been used to investigate the role of multi-histidinic and glutamate sites in coordinating metal ions. (1)H-(1)H TOCSY, (1)H-(13)C HSQC multidimensional NMR techniques were performed to understand the details of metal binding sites and the conformational behaviour of the peptide. The effects of the peptide titration with the two metals have been followed by paramagnetic selective line-broadening in the 1D NMR spectra and the signals' disappearance in the 2D (1)H-(13)C HSQC and (1)H-(1)H TOCSY. Both ions showed common binding donor atoms: the main manganese and cobalt binding centre of the peptide fragment is associated with histidine and glutamate residues. The specific perturbation of NMR resonances indicated that the coordination involves imidazole Nε of histidine and carboxyl γ-O of glutamate residue. All the three imidazole Nε of His6, His16 and His26, as well as carboxyl γ-O of Glu9, Glu19 and Glu29, in an octahedral arrangement are involved in the coordination in the physiological pH range. The involvement of hydroxyl γ-O from the threonine (or serine) side chain can also be observed. Manganese and cobalt complexation induces important structural changes within the C-terminal portion of the ligand, constraining it to leave its disordered conformation. A model of the structure of manganese and cobalt species can be obtained from our data.

  13. Histidine in Continuum Electrostatics Protonation State Calculations

    Science.gov (United States)

    Couch, Vernon; Stuchebruckhov, Alexei

    2014-01-01

    A modification to the standard continuum electrostatics approach to calculate protein pKas which allows for the decoupling of histidine tautomers within a two state model is presented. Histidine with four intrinsically coupled protonation states cannot be easily incorporated into a two state formalism because the interaction between the two protonatable sites of the imidazole ring is not purely electrostatic. The presented treatment, based on a single approximation of the interrelation between histidine’s charge states, allows for a natural separation of the two protonatable sites associated with the imidazole ring as well as the inclusion of all protonation states within the calculation. PMID:22072521

  14. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases

    Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based

  15. 21 CFR 582.5361 - Histidine.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  16. Modulating short tryptophan- and arginine-rich peptides activity by substitution with histidine.

    Science.gov (United States)

    Bacalum, Mihaela; Janosi, Lorant; Zorila, Florina; Tepes, Ana-Maria; Ionescu, Cristina; Bogdan, Elena; Hadade, Niculina; Craciun, Liviu; Grosu, Ion; Turcu, Ioan; Radu, Mihai

    2017-07-01

    High antimicrobial efficacy of short tryptophan-and arginine-rich peptides makes them good candidates in the fight against pathogens. Substitution of tryptophan and arginine by histidine could be used to modulate the peptides efficacy by optimizing their structures. The peptide (RRWWRWWRR), reported to showed good antimicrobial efficacy, was used as template, seven new analogs being designed substituting tryptophan or arginine with histidine. The peptides' efficacy was tested against E. coli, B. subtilis and S. aureus. The cytotoxicity and hemolytic effect were evaluated and the therapeutic index was inferred for each peptide. Atomic force microscopy and molecular simulation were used to analyze the effects of peptides on bacterial membrane. The substitution of tryptophan by histidine proved to strongly modulate the antimicrobial activity, mainly by changing the peptide-to-membrane binding energy. The substitution of arginine has low effect on the antimicrobial efficacy. The presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides in some cases maintaining the same efficacy against bacteria. The peptides' antimicrobial activity was correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter. The results show that some of these peptides have the potential to become good candidates to fight against bacteria. The substitution by histidine proved to fine tune the therapeutic index allowing the optimization of the peptide structure mainly by changing its binding energy and 3D-hydrophobic moment. The short tryptophan reach peptides therapeutic index can be maximized using the histidine substitution to optimize their structure. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Effect of dietary electrolytes and histidine on histidine metabolism and acid-base balance in rainbow trout (Salmo gairdneri)

    Science.gov (United States)

    Chiu, Y.N.; Austic, R.E.; Rumsey, G.L.

    1984-01-01

    1. Rainbow trout fingerlings were fed diets containing 1.2, 1.8 and 2.6% histidine and two mixtures of Na, K and Cl (Na + K - Cl = 0 or -200 meq/kgdiet) in a factorial design.2. Growth and efficiency of feed conversion were not affected by histidine in the diet when it contained the −200 meq/kg electrolyte mixture, but with the 0 meq/kg level, 2.6% histidine depressed both measures of response.3. Histidine increased plasma and muscle histidine levels, increased hepatic histidase activity, but did not affect hepatic histidine-pyruvate aminotransferase activity.4. Muscle-free histidine concentrations were markedly higher and lysine concentrations were lower in trout receiving 0 meq/kg than those receiving the −200 meq/kg electrolyte mixture.5. The electrolyte balance of the diet has a marked effect on the metabolism of histidine in trout.

  18. Inhibition of Transcription of the Histidine Operon In Vitro by the First Enzyme of the Histidine Pathway

    Science.gov (United States)

    Blasi, Francesco; Bruni, Carmelo B.; Avitabile, Alessandra; Deeley, Roger G.; Goldberger, Robert F.; Meyers, Marilyn M.

    1973-01-01

    An in vitro system was developed for transcription of the histidine operon of Esherichia coli carried in the genome of a defective ϕ80 transducing phage. The messenger RNA (mRNA) of the histidine operon synthesized in the in vitro system was detected by hybridization to single strands of both ϕ80 and ϕ80dhis DNA, and by competition of this hybridization with unlabeled histidine mRNA that had been synthesized in vivo (RNA extracted from cells in which the histidine operon had been derepressed). Under the conditions used, RNA complementary to the histidine operon was about 15% of the total RNA that was synthesized in vitro from the ϕ80dhis DNA template. The RNA complementary to the histidine operon was synthesized on the “sense” strand (the R strand) of ϕ80dhis in the form of a polycistronic message with a sedimentation coefficient (about 38 S) very close to that observed for the histidine mRNA synthesized in vivo. Synthesis of the histidine operon RNA appears to be subject to control in vitro. Addition of the first enzyme of the pathway for histidine biosynthesis blocked transcription of the histidine operon specifically, strongly suggesting that this enzyme acts as a regulatory protein for the histidine operon. PMID:4582195

  19. Neighbor-directed Histidine N(τ)–Alkylation: A Route to Imidazolium-containing Phosphopeptide Macrocycles

    Science.gov (United States)

    Qian, Wen-Jian; Park, Jung-Eun; Grant, Robert; Lai, Christopher C.; Kelley, James A.; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.

    2016-01-01

    Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In our current work, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts. PMID:26152807

  20. Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase.

    Science.gov (United States)

    Liyasova, Mariya S; Schopfer, Lawrence M; Lockridge, Oksana

    2013-03-25

    Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Identification of a histidine acid phosphatase (phyA)-like gene in Arabidopsis thaliana.

    Science.gov (United States)

    Mullaney, E J; Ullah, A H

    1998-10-09

    A close examination of the protein sequence encoded by the Arabidopsis thaliana gene F21M12.26 reveals the gene product to be a phosphomonoesterase, acid optimum (EC 3.1.3.2). A subclass of this broad acid phosphatase is also known as 'histidine acid phosphatase. ' This is the first sequence-based evidence for a 'histidine acid phosphatase' in a dicotyledon. One important member of this class of enzymes is Aspergillus niger (ficuum) phytase, which came into prominence for its commercial application as a feed additive. The putative protein from A. thaliana gene F21M12.26 shares many important features of Aspergillus phytase, namely, size, active-site sequence, catalytic dipeptide and ten cysteine residues located in the key areas of the molecule, but lacks all nine N-glycosylation sites. Copyright 1998 Academic Press.

  2. A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches.

    Science.gov (United States)

    Murtaugh, Megan L; Fanning, Sean W; Sharma, Tressa M; Terry, Alexandra M; Horn, James R

    2011-09-01

    There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK(a) change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine "hot-spots," which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications. Copyright © 2011 The Protein Society.

  3. Influence of histidine incorporation on buffer capacity and gene transfection efficiency of HPMA-co-oligolysine brush polymers.

    Science.gov (United States)

    Shi, Julie; Schellinger, Joan G; Johnson, Russell N; Choi, Jennifer L; Chou, Brian; Anghel, Ersilia L; Pun, Suzie H

    2013-06-10

    One of the major intracellular barriers to nonviral gene delivery is efficient endosomal escape. The incorporation of histidine residues into polymeric constructs has been found to increase endosomal escape via the proton sponge effect. Statistical and diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), oligolysine, and oligohistidine were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization and tested for in vitro transfection efficiency, buffering ability, and polyplex uptake mechanism via the use of chemical endocytic inhibitors. Interestingly, histidine-containing statistical and diblock polymers exhibited increased buffer capacity in different endosomal pH ranges. Statistical copolymers transfected better than block copolymers that contained similar amounts of histidine. In addition, only the polymer containing the highest incorporation of oligohistidine residues led to increases in transfection efficiency over the HPMA-oligolysine base polymer. Thus, for these polymer architectures, high histidine incorporation may be required for efficient endosomal escape. Furthermore, inhibitor studies indicate that nonacidified caveolae-mediated endocytosis may be the primary route of transfection for these copolymers, suggesting that alternative approaches for increasing endosomal escape may be beneficial for enhancing transfection efficiency with these HPMA-oligolysine copolymers.

  4. Electron Transfer from Azide Radical to Histidine Generates ...

    African Journals Online (AJOL)

    The formation of histidinyl radical (HR), which is a product of electron transfer reaction between histidine and some free radicals, was studied by pulse radiolysis. The reaction between histidine and azide radicals was found to produce HR, which has a distinct absorption spectrum with peaks at 300, 480 and 520 nm.

  5. Does aluminium bind to histidine? An NMR investigation of amyloid β12 and amyloid β16 fragments.

    Science.gov (United States)

    Narayan, Priya; Krishnarjuna, Bankala; Vishwanathan, Vinaya; Jagadeesh Kumar, Dasappa; Babu, Sudhir; Ramanathan, Krishna Venkatachala; Easwaran, Kalpathy Ramaier Katchap; Nagendra, Holenarasipur Gundurao; Raghothama, Srinivasarao

    2013-07-01

    Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N-terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C-terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium-binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal-binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets. © 2013 John Wiley & Sons A/S.

  6. GENERATION OF A PROTON MOTIVE FORCE BY HISTIDINE DECARBOXYLATION AND ELECTROGENIC HISTIDINE HISTAMINE ANTIPORT IN LACTOBACILLUS-BUCHNERI

    NARCIS (Netherlands)

    MOLENAAR, D; BOSSCHER, JS; TENBRINK, B; DRIESSEN, AJM; KONINGS, WN

    Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (DELTApsi), inside negative, upon

  7. Functional Divergence of Poplar Histidine-Aspartate Kinase HK1 Paralogs in Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    François Héricourt

    2016-12-01

    Full Text Available Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

  8. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    Directory of Open Access Journals (Sweden)

    Maria Ranieri-Raggi

    2014-05-01

    Full Text Available Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD and the metal binding protein histidine-proline-rich glycoprotein (HPRG acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua(μ-carboxylatodizinc(II core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

  9. Metal-mediated molecular self-healing in histidine-rich mussel peptides.

    Science.gov (United States)

    Schmidt, Stephan; Reinecke, Antje; Wojcik, Felix; Pussak, Daniel; Hartmann, Laura; Harrington, Matthew James

    2014-05-12

    Mussels withstand high-energy wave impacts in rocky seashore habitats by fastening tightly to surfaces with tough and self-healing proteinaceous fibers called byssal threads. Thread mechanical behavior is believed to arise from reversibly breakable metal coordination cross-links embedded in histidine-rich protein domains (HRDs) in the principle load-bearing proteins comprising the fibrous thread core. In order to investigate HRD behavior at the molecular level, we have synthesized a histidine-rich peptide derived from mussel proteins (His5-bys) and studied its reversible adhesive self-interaction in the presence and absence of metal ions using PEG-based soft-colloidal probes (SCPs). Adhesion energies of greater than 0.3 mJ/m(2) were measured in the presence of metal ions, and the stiffness of the modified SCPs exhibited a 3-fold increase, whereas no adhesion was observed in the absence of metals. Raman spectroscopy confirmed the presence of metal-coordination via histidine residues by the peptide-supporting the role of His-metal complexes in the mechanical behavior of the byssus.

  10. Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

    NARCIS (Netherlands)

    Bem, A.E.; Velikova, N.R.; Pellicer, M.T.; Baarlen, van P.; Marina, A.; Wells, J.M.

    2015-01-01

    Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In

  11. Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

    Science.gov (United States)

    Agarwal, Pragati; Singh, Jyoti; Singh, R P

    2017-05-01

    Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

  12. Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

    Science.gov (United States)

    Stratton, Amanda; Ericksen, Matthew; Harris, Travis V; Symmonds, Nick; Silverstein, Todd P

    2017-02-01

    The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his40 and his57 ) quickly and completely, with an IC50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation. © 2017 The Protein Society.

  13. A Rhizobium radiobacter Histidine Kinase Can Employ Both Boolean AND and OR Logic Gates to Initiate Pathogenesis.

    Science.gov (United States)

    Fang, Fang; Lin, Yi-Han; Pierce, B Daniel; Lynn, David G

    2015-10-12

    The molecular logic gates that regulate gene circuits are necessarily intricate and highly regulated, particularly in the critical commitments necessary for pathogenesis. We now report simple AND and OR logic gates to be accessible within a single protein receptor. Pathogenesis by the bacterium Rhizobium radiobacter is mediated by a single histidine kinase, VirA, which processes multiple small molecule host signals (phenol and sugar). Mutagenesis analyses converged on a single signal integration node, and finer functional analyses revealed that a single residue could switch VirA from a functional AND logic gate to an OR gate where each of two signals activate independently. Host range preferences among natural strains of R. radiobacter correlate with these gate logic strategies. Although the precise mechanism for the signal integration node requires further analyses, long-range signal transmission through this histidine kinase can now be exploited for synthetic signaling circuits. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Electrophilic catalysis in triosephosphate isomerase: The role of histidine-95

    Energy Technology Data Exchange (ETDEWEB)

    Komives, E.A.; Chang, L.C.; Knowles, J.R. (Harvard Univ., Cambridge, MA (USA)); Lolis, E.; Tilton, R.F.; Petsko, G.A. (Massachusetts Inst. of Tech., Cambridge, MA (USA))

    1991-03-26

    Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be lower than that of dihydroxyacetone phosphate free in solution, and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1,732 and 1,742 cm{sup {minus}1}. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-{angstrom} resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.

  15. Extracellular guanosine regulates extracellular adenosine levels

    Science.gov (United States)

    Cheng, Dongmei; Jackson, Travis C.; Verrier, Jonathan D.; Gillespie, Delbert G.

    2013-01-01

    The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)−6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases

  16. Menkes disease and response to copper histidine: An Indian case series

    Directory of Open Access Journals (Sweden)

    Sangeetha Yoganathan

    2017-01-01

    Full Text Available Background: Menkes disease (MD is an X-linked recessive neurodegenerative disorder caused by mutations in ATP7A gene. Depending on the residual ATP7A activity, manifestation may be classical MD, occipital horn syndrome, or distal motor neuropathy. Neurological sparing is expected in female carriers. However, on rare occasions, females may manifest with classical clinical phenotype due to skewed X-chromosome inactivation, X-autosome translocation, and XO genotype. Here, we describe a small series of probands with MD and their response to copper histidine therapy. This series also includes a female with X-13 translocation manifesting neurological symptoms. Methods: The clinical profile, laboratory and radiological data, and follow-up of four children with MD were collected from the hospital database and are being presented. Results: All the four children in our series had developmental delay, recurrent respiratory tract infections, hair and skeletal changes, axial hypotonia, tortuous vessels on imaging, low serum copper, ceruloplasmin, and elevated lactate. Fetal hypokinesia and fetal growth retardation were present in two cases. Failure to thrive was present in three children and only one child had epilepsy. Subcutaneous copper histidine was administered to all children. The average time lapse in the initiation of treatment was 20.3 months, and average duration of follow-up was 14.3 months. Conclusion: We conclude that copper histidine therapy is beneficial in reversing the skin and hair changes, improving appendicular tone, socio-cognitive milestones, and improving weight gain, and immunity. Early diagnosis and management of MD are essential to have a better clinical outcome. More research is needed to explore and devise new strategies in the management of patients with MD.

  17. Comparison of fractal dimension and Shannon entropy in myocytes from rats treated with histidine-tryptophan-glutamate and histidine-tryptophan cetoglutarate

    Science.gov (United States)

    de Oliveira, Marcos Aurélio Barboza; Brandi, Antônio Carlos; dos Santos, Carlos Alberto; Botelho, Paulo Henrique Husseni; Cortez, José Luís Lasso; de Godoy, Moacir Fernandes; Braile, Domingo Marcolino

    2014-01-01

    Introduction Solutions that cause elective cardiac arrest are constantly evolving, but the ideal compound has not yet been found. The authors compare a new cardioplegic solution with histidine-tryptophan-glutamate (Group 2) and other one with histidine-tryptophan-cetoglutarate (Group 1) in a model of isolated rat heart. Objective To quantify the fractal dimension and Shannon entropy in rat myocytes subjected to cardioplegia solution using histidine-tryptophan with glutamate in an experimental model, considering the caspase markers, IL-8 and KI-67. Methods Twenty male Wistar rats were anesthetized and heparinized. The chest was opened, the heart was withdrawn and 40 ml/kg of cardioplegia (with histidine-tryptophan-cetoglutarate or histidine-tryptophan-glutamate solution) was infused. The hearts were kept for 2 hours at 4ºC in the same solution, and thereafter placed in the Langendorff apparatus for 30 min with Ringer-Locke solution. Analyzes were performed for immunohistochemical caspase, IL-8 and KI-67. Results The fractal dimension and Shannon entropy were not different between groups histidine-tryptophan-glutamate and histidine-tryptophan-acetoglutarate. Conclusion The amount of information measured by Shannon entropy and the distribution thereof (given by fractal dimension) of the slices treated with histidine-tryptophan-cetoglutarate and histidine-tryptophan-glutamate were not different, showing that the histidine-tryptophan-glutamate solution is as good as histidine-tryptophan-acetoglutarate to preserve myocytes in isolated rat heart. PMID:25140464

  18. The question of histidine content in c-type cytochromes.

    Science.gov (United States)

    Cusanovich, M A; Meyer, T; Tedro, S M; Kamen, M D

    1971-03-01

    Reports that histidine may not occur in heme peptides derived from c-type cytochromes isolated from chloroplasts of Euglena gracilis and Porphyra sp. have not been substantiated in the present investigation, in which the amino acid composition and a partial sequence were determined for a heme peptide derived from the c-type cytochromes of a strain of Euglena closely related to that used in the previous studies. It is concluded that no evidence exists to challenge the generalization that histidine is always present vicinal to the hemebinding site in c-type cytochromes.

  19. Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Varsha C. Mohanan

    2017-05-01

    Full Text Available Two-component signal transduction (TCST pathways play crucial roles in many cellular functions such as stress responses, biofilm formation, and sporulation. The histidine phosphotransferase (HPt, which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s, and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK or the downstream response regulators (RR in two-component system, the HPts have not been well-studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

  20. Fungal histidine phosphotransferase plays a crucial role in photomorphogenesis and pathogenesis in Magnaporthe oryzae

    Science.gov (United States)

    Mohanan, Varsha C.; Chandarana, Pinal M.; Chattoo, Bharat. B.; Patkar, Rajesh N.; Manjrekar, Johannes

    2017-05-01

    Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

  1. Safety, absorption, and antioxidant effects of chromium histidine

    Science.gov (United States)

    Supplemental chromium has been shown to be involved in the alleviation of the metabolic syndrome, glucose intolerance, polycystic ovary syndrome, depression, excess body fat, and gestational, steroid-induced, and type 2 diabetes. Chromium amino acid complexes that contained histidine displayed cons...

  2. 21 CFR 862.1375 - Histidine test system.

    Science.gov (United States)

    2010-04-01

    ... often resulting in mental retardation and disordered speech development. (b) Classification. Class I... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Histidine test system. 862.1375 Section 862.1375...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

  3. Hydrogen peroxide induce modifications of human extracellular superoxide dismutase that results in enzyme inhibition

    Directory of Open Access Journals (Sweden)

    Randi H. Gottfredsen

    2013-01-01

    Full Text Available Superoxide dismutase (EC-SOD controls the level of superoxide in the extracellular space by catalyzing the dismutation of superoxide into hydrogen peroxide and molecular oxygen. In addition, the enzyme reacts with hydrogen peroxide in a peroxidase reaction which is known to disrupt enzymatic activity. Here, we show that the peroxidase reaction supports a site-specific bond cleavage. Analyses by peptide mapping and mass spectrometry shows that oxidation of Pro112 supports the cleavage of the Pro112–His113 peptide bond. Substitution of Ala for Pro112 did not inhibit fragmentation, indicating that the oxidative fragmentation at this position is dictated by spatial organization and not by side-chain specificity. The major part of EC-SOD inhibited by the peroxidase reaction was not fragmented but found to encompass oxidations of histidine residues involved in the coordination of copper (His98 and His163. These oxidations are likely to support the dissociation of copper from the active site and thus loss of enzymatic activity. Homologous modifications have also been described for the intracellular isozyme, Cu/Zn-SOD, reflecting the almost identical structures of the active site within these enzymes. We speculate that the inactivation of EC-SOD by peroxidase activity plays a role in regulating SOD activity in vivo, as even low levels of superoxide will allow for the peroxidase reaction to occur.

  4. Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

    Science.gov (United States)

    Bergeron, Eric; Basak, Ajoy; Decroly, Etienne; Seidah, Nabil G

    2003-07-15

    The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.

  5. Neighbor-Directed Histidine N (s)–Alkylation: A Route to Imidazolium-Containing Phosphopeptide Macrocycles-Biopolymers | Center for Cancer Research

    Science.gov (United States)

    Our recently discovered, selective, on-resin route to N(s)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation.

  6. Effect of Phosphorylation on Hydrogen-Bonding Interactions of the Active Site Histidine of the Phosphocarrier Protein HPr of the Phosphoenolpyruvate-Dependent Phosphotransferase System Determined by 15N NMR Spectroscopy

    NARCIS (Netherlands)

    Dijk, Alard A. van; Lange, Liesbeth C.M. de; Bachovchin, William W.; Robillard, George T.

    1990-01-01

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this

  7. Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

    Science.gov (United States)

    Sen, Hrishiraj; Aggarwal, Nikhil; Ishionwu, Chibueze; Hussain, Nosheen; Parmar, Chandni; Jamshad, Mohammed; Bavro, Vassiliy N; Lund, Peter A

    2017-09-15

    The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this. IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps

  8. Contributions of the Histidine Side Chain and the N-terminal α-Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH

    Science.gov (United States)

    Ballin, Jeff D.; Prevas, James P.; Ross, Christina R.; Toth, Eric A.; Wilson, Gerald M.; Record, M. Thomas

    2010-01-01

    Interactions of histidine with nucleic acid phosphates and histidine pKa shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK (+NH3-His-Trp-Lys-Lys-NH2) and its lysine analog KWKK (+NH3-Lys-Trp-Lys-Lys-NH2) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, Kobs, and magnitudes of log-log salt derivatives SKobs ≡ (∂logKobs/∂log[Na+]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and α-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same Kobs and SKobs within uncertainty, and these SKobs values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pKa shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pKa shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the α-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific versus nonspecific nucleic acid binding. PMID:20108951

  9. Copper Complexes Of Di-, Tri-, And Tetra-Peptides Containing Tryptophan, Histidine And Arginine

    OpenAIRE

    El Naggar, A. M. [احمد محمد النجار; El-Ghaffar, S. A. A.; Zaher, M. R.

    1983-01-01

    Fifty Seven copper complexes of di-, tri-. and tetra-peptides containing tryptophan, histidine and arginine are studied spectrophotometrically. The ^a, and colour of the complexes are dependent on the sequence of the amino acid in the dipeptide methyl esters of tryptophan and arginine; and independent on the sequence of dipeptides of histidine or in any of the tri- and tetra-peptides of histidine, arginine and tryptophan. The results achieved confirmed that the nitrogen atoms of the indole nu...

  10. Low-temperature Raman spectra of L-histidine crystal

    Energy Technology Data Exchange (ETDEWEB)

    Souda, G.P. de; Freire, P.T.C.; Mendes Filho, J.; Melo, F.E.A., E-mail: tarso@fisica.ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Fisica; Lima, C.L. [Universidade Federal do Piaui (UFPI), Teresina, PI (Brazil). Departamento de Fisica

    2013-07-01

    We present a Raman spectroscopy investigation of the vibrational properties of l-histidine crystals at low temperatures. The temperature dependence of the spectra show discontinuities at 165 K, which we identify with modifications in the bonds associated to both the NH{sub 3}{sup +} and CO{sub 2} − motifs indicative of a conformational phase transition that changes the intermolecular bonds. Additional evidence of such a phase transition is provided by differential scanning calorimetry measurements, which identified an enthalpic anomaly at ∼165 K. (author)

  11. Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

    Science.gov (United States)

    Rice, Selena L; Preimesberger, Matthew R; Johnson, Eric A; Lecomte, Juliette T J

    2014-12-01

    The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Cloning and sequencing of the histidine decarboxylase gene from Photobacterium phosphoreum and its functional expression in Escherichia coli.

    Science.gov (United States)

    Morii, Hideaki; Kasama, Kentaro; Herrera-Espinoza, Raul

    2006-08-01

    The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5'-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.

  13. The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

    Science.gov (United States)

    Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena

    2017-10-20

    The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

  14. Critical amino acid residues involved in the electrogenic sodium–bicarbonate cotransporter kNBC1-mediated transport

    Science.gov (United States)

    Abuladze, Natalia; Azimov, Rustam; Newman, Debra; Sassani, Pakan; Liu, Weixin; Tatishchev, Sergei; Pushkin, Alexander; Kurtz, Ira

    2005-01-01

    We have previously reported a topological model of the electrogenic Na+–HCO3− cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na+-dependent base flux of the mutants was determined using intracellular pH measurements with 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na+-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented. PMID:15817634

  15. Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornata

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zuxu; de Serrano, Vesna; Betts, Laurie; Franzen, Stefan; (NCSU); (UNC)

    2009-01-28

    The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5 {angstrom} away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25 {angstrom} from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP.

  16. In Silico Characterization of Histidine Acid Phytase Sequences

    Directory of Open Access Journals (Sweden)

    Vinod Kumar

    2012-01-01

    Full Text Available Histidine acid phytases (HAPhy are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA, homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal “RHGXRXP” and C-terminal “HD.” Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 “SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF” was present in 38 protein sequences representing clusters 1 (PhyA and 2 (PhyB. Cluster 3 (AppA contains motif 9 “KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP” as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. This in silico analysis might contribute in the classification and future genetic engineering of this most diverse class of phytase.

  17. Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

    Science.gov (United States)

    Moya-García, Aurelio A; Rodríguez-Agudo, Daniel; Hayashi, Hideyuki; Medina, Miguel Angel; Urdiales, José Luis; Sánchez-Jiménez, Francisca

    2011-06-14

    Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases.

  18. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    Science.gov (United States)

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Feeding filaggrin: effects of l-histidine supplementation in atopic dermatitis.

    Science.gov (United States)

    Tan, Siao Pei; Brown, Simon B; Griffiths, Christopher Em; Weller, Richard B; Gibbs, Neil K

    2017-01-01

    Atopic dermatitis (AD), also known as eczema, is one of the most common chronic skin conditions worldwide, affecting up to 16% of children and 10% of adults. It is incurable and has significant psychosocial and economic impacts on the affected individuals. AD etiology has been linked to deficiencies in the skin barrier protein, filaggrin. In mammalian skin, l-histidine is rapidly incorporated into filaggrin. Subsequent filaggrin proteolysis releases l-histidine as an important natural moisturizing factor (NMF). In vitro studies were conducted to investigate the influence of l-histidine on filaggrin processing and barrier function in human skin-equivalent models. Our further aim was to examine the effects of daily oral l-histidine supplementation on disease severity in adult AD patients. We conducted a randomized, double-blind, placebo-controlled, crossover, nutritional supplementation pilot study to explore the effects of oral l-histidine in adult AD patients (n=24). In vitro studies demonstrated that l-histidine significantly increased both filaggrin formation and skin barrier function (Pl-histidine significantly reduced (P0.32). The clinical effect of oral l-histidine in AD was similar to that of mid-potency topical corticosteroids and combined with its safety profile suggests that it may be a safe, nonsteroidal approach suitable for long-term use in skin conditions that are associated with filaggrin deficits such as AD.

  20. Synthesis and catalytic activity of histidine-based NHC ruthenium complexes.

    Science.gov (United States)

    Monney, Angèle; Venkatachalam, Galmari; Albrecht, Martin

    2011-03-28

    Main-chain C,N-protected histidine has been successfully alkylated at both side-chain nitrogens. The corresponding histidinium salt was metallated with ruthenium(II) by a transmetalation procedure, thus providing histidine-derived NHC ruthenium complexes. These bio-inspired complexes show appreciable activity in the catalytic transfer hydrogenation of ketones.

  1. Synthesis of selectively labeled histidine and its methylderivatives with deuterium, tritium, and carbon-14.

    Science.gov (United States)

    Šamonina-Kosicka, J; Kańska, M

    2013-05-30

    Isotopologues of l-histidine and its N-methylderivatives labeled with deuterium and tritium at the 5-position in the imidazole ring were obtained using the isotope exchange method. The deuterium-labeled isotopologues [5-(2)H]-l-histidine, [5-(2)H]-N(τ) -methyl-l-histidine, [5-(2)H]-N(π) -methyl-l-histidine, and [2,5-(2)H(2)]-l-histidine were synthesized by isotope exchange method carried out in a fully deuterated medium with. The same reaction conditions were applied to synthesize [5-(3)H]-N(τ) -methyl-l-histidine, [5-(3)H]-N(π) -methyl-l-histidine, and [5-(3)H]-l-histidine with specific activity of 2.0, 5.0, and 2.6 MBq/mmol, respectively. The N(π) -[methyl-(14)C]-histamine was obtained with specific activity of 0.23 MBq/mmol in a one-step reaction by the direct methylation of histamine by [(14)C]iodomethane. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Rational Design of Selective Adenine-Based Scaffolds for Inactivation of Bacterial Histidine Kinases.

    Science.gov (United States)

    Goswami, Manibarsha; Wilke, Kaelyn E; Carlson, Erin E

    2017-10-12

    Bacterial histidine kinases (HKs) are quintessential regulatory enzymes found ubiquitously in bacteria. Apart from their regulatory roles, they are also involved in the production of virulence factors and conferring resistance to various antibiotics in pathogenic microbes. We have previously reported compounds that inhibit multiple HKs by targeting the conserved catalytic and ATP-binding (CA) domain. Herein, we conduct a detailed structure-activity relationship assessment of adenine-based inhibitors using biochemical and docking methods. These studies have resulted in several observations. First, interaction of an inhibitor's amine group with the conserved active-site Asp is essential for activity and likely dictates its orientation in the binding pocket. Second, a N-NH-N triad in the inhibitor scaffold is highly preferred for binding to conserved Gly:Asp:Asn residues. Lastly, hydrophobic electron-withdrawing groups at several positions in the adenine core enhance potency. The selectivity of these inhibitors was tested against heat shock protein 90 (HSP90), which possesses a similar ATP-binding fold. We found that groups that target the ATP-lid portion of the catalytic domain, such as a six-membered ring, confer selectivity for HKs.

  3. Syntheses of stable, synthetic diadenosine polyphosphate analogues using recombinant histidine-tagged lysyl tRNA synthetase (LysU).

    Science.gov (United States)

    Wright, Michael; Azhar, M Ameruddin; Kamal, Ahmed; Miller, Andrew D

    2014-05-15

    Recombinant Escherichia coli lysyl-tRNA synthase (LysU) has been previously utilised in the production of stabile, synthetic diadenosine polyphosphate (ApnA) analogues. Here we report on the extended use of a new recombinant histidine residue-tagged LysU as a tool for highly controlled phosphatephosphate bond formation between nucleotides, avoiding the need for complex protecting group chemistries. Resulting high yielding tandem LysU-based biosynthetic-synthetic/synthetic-biosynthetic strategies emerge for the preparation of varieties of ApnA analogues directly from inexpensive natural nucleotides and nucleosides. Analogues so formed make a useful small library with which to probe ApnA activities in vitro and in vivo leading to the discovery of new, potentially potent biopharmaceuticals active against chronic pain and other chronic, high-burden disease states. Copyright © 2014. Published by Elsevier Ltd.

  4. Effects of intraperitoneally administered L-histidine on food intake, taste, and visceral sensation in rats.

    Science.gov (United States)

    Okusha, Yuka; Hirai, Yoshiyuki; Maezawa, Hitoshi; Hisadome, Kazunari; Inoue, Nobuo; Yamazaki, Yutaka; Funahashi, Makoto

    2017-07-01

    To evaluate relative factors for anorectic effects of L-histidine, we performed behavioral experiments for measuring food and fluid intake, conditioned taste aversion (CTA), taste disturbance, and c-Fos immunoreactive (Fos-ir) cells before and after i.p. injection with L-histidine in rats. Animals were injected with saline (9 ml/kg, i.p.) for a control group, and saline (9 ml/kg, i.p.) containing L-histidine (0.75, 1.5, 2.0 g/kg) for a L-histidine group. Injection of L-histidine decreased the average value of food intake, and statistically significant anorectic effects were found in animals injected with 1.5 or 2.0 g/kg L-histidine but not with 0.75 g/kg L-histidine. Taste abnormalities were not detected in any of the groups. Animals injected with 2.0 g/kg L-histidine were revealed to present with nausea by the measurement of CTA. In this group, a significant increase in the number of Fos-ir cells was detected both in the area postrema and the nucleus tractus solitarius (NTS). In the 0.75 g/kg L-histidine group, a significant increase in the number of Fos-ir cells was detected only in the NTS. When the ventral gastric branch vagotomy was performed, recovery from anorexia became faster than the sham-operated group, however, vagotomized rats injected with 2.0 g/kg L-histidine still acquired CTA. These data indicate that acute anorectic effects induced by highly concentrated L-histidine are partly caused by induction of nausea and/or visceral discomfort accompanied by neuronal activities in the NTS and the area postrema. We suggest that acute and potent effects of L-histidine on food intake require substantial amount of L-histidine in the diet.

  5. The effect of an adding histidine on biological activity and stability of Pc-pis from Pseudosciaena crocea.

    Science.gov (United States)

    Mao, Yong; Niu, Sufang; Xu, Xin; Wang, Jun; Su, Yongquan; Wu, Yang; Zhong, Shengping

    2013-01-01

    Pc-pis is a novel piscidin-like antimicrobial polypeptide that was identified in Pseudosciaena crocea. Although active against most bacteria tested, Pc-pis was inactive against Aeromonas hydrophila and Pseudomonas aeruginosa. The Pc-pis analogue Pc-pis-His was designed by adding a histidine residue at the carboxyl terminal. Pc-pis-His demonstrated a more broad-spectrum and stronger antimicrobial activity against a representative set of microorganisms and more potent antiparasitic activity against Cryptocaryon irritans trophonts than Pc-pis. The stability assay revealed that Pc-pis-His was active against Staphylococcus aureus not only in acidic (pH 5.5-7.3) and relatively low concentration monovalent cation (0-160 mM NaCl) environments but also in alkaline (pH 7.5-9.5), divalent cation (1.25-160 mM MgCl2 and 1.25-40 mM CaCl2) and high concentration monovalent cation (320-2560 mM NaCl) environments, which indicates that the added histidine residue conferred better salt-, acid- and alkali-tolerance to Pc-pis-His. Pc-pis-His also possessed the desired heat-tolerance, which was reflected by the antimicrobial activity of the peptide after being boiled for 10-60 minutes. Hemolytic activity analysis revealed that Pc-pis-His at concentrations up to 6 µM exhibited no hemolysis against human erythrocytes, with 6 µM being a concentration that is highly active against most of the microorganisms tested, although the hemolytic activity of Pc-pis-His was enhanced compared to Pc-pis. These results provide a unique, reasonable basis for designing novel piscidins with potent, broad-spectrum and stable antimicrobial activity and new insight into the future development of piscidins as potential therapeutic agents against microbial and external protozoan parasite infections.

  6. The effect of an adding histidine on biological activity and stability of Pc-pis from Pseudosciaena crocea.

    Directory of Open Access Journals (Sweden)

    Yong Mao

    Full Text Available Pc-pis is a novel piscidin-like antimicrobial polypeptide that was identified in Pseudosciaena crocea. Although active against most bacteria tested, Pc-pis was inactive against Aeromonas hydrophila and Pseudomonas aeruginosa. The Pc-pis analogue Pc-pis-His was designed by adding a histidine residue at the carboxyl terminal. Pc-pis-His demonstrated a more broad-spectrum and stronger antimicrobial activity against a representative set of microorganisms and more potent antiparasitic activity against Cryptocaryon irritans trophonts than Pc-pis. The stability assay revealed that Pc-pis-His was active against Staphylococcus aureus not only in acidic (pH 5.5-7.3 and relatively low concentration monovalent cation (0-160 mM NaCl environments but also in alkaline (pH 7.5-9.5, divalent cation (1.25-160 mM MgCl2 and 1.25-40 mM CaCl2 and high concentration monovalent cation (320-2560 mM NaCl environments, which indicates that the added histidine residue conferred better salt-, acid- and alkali-tolerance to Pc-pis-His. Pc-pis-His also possessed the desired heat-tolerance, which was reflected by the antimicrobial activity of the peptide after being boiled for 10-60 minutes. Hemolytic activity analysis revealed that Pc-pis-His at concentrations up to 6 µM exhibited no hemolysis against human erythrocytes, with 6 µM being a concentration that is highly active against most of the microorganisms tested, although the hemolytic activity of Pc-pis-His was enhanced compared to Pc-pis. These results provide a unique, reasonable basis for designing novel piscidins with potent, broad-spectrum and stable antimicrobial activity and new insight into the future development of piscidins as potential therapeutic agents against microbial and external protozoan parasite infections.

  7. Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping.

    Science.gov (United States)

    Rehman, Ateeq Ur; Cser, Krisztián; Sass, László; Vass, Imre

    2013-06-01

    Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of (1)O2, and suppressed by the (1)O2 quencher NaN3. Due to the limited mobility of (1)O2 these data demonstrate that exogenous histidine reaches close vicinity of (1)O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that (1)O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied (1)O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518-8527; Cser and Vass 2007, BBA 1767:233-243). We found that the D1-Gln130Glu mutant showed decreased (1)O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both (1)O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which (3)P680 mediated (1)O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Oral administration of vitamin C and histidine attenuate cyclophosphamide-induced hemorrhagic cystitis in rats.

    Science.gov (United States)

    Farshid, Amir Abbas; Tamaddonfard, Esmaeal; Ranjbar, Sepideh

    2013-01-01

    Cyclophosphamide (CP), a widely used antineoplastic drug causes hemorrhagic cystitis (HC) mainly via induction of oxidative stress. Both vitamin C and histidine have antioxidant properties. The present study aimed to investigate the effects of oral (p.o.) administration of vitamin C and histidine on the CP-induced HC in rats. The animals were divided into two major groups I and II with four subgroups (a, b, c, and d) in each. Groups I and II were treated with intraperitoneal (i.p.) injections of normal saline and CP (200 mg/kg), respectively, thereafter, normal saline, vitamin C (200 mg/kg), histidine (200 mg/kg) and vitamin C plus histidine were p.o. administered in subgroups a, b, c, and d, respectively, three times (2, 6, and 24 h) after i.p. injections of normal saline and CP. Blood samples were assayed for total antioxidant capacity (TAC) and malondialdehyde (MDA) levels. Histopathological changes of bladder wall were investigated. The decreased TAC and increased MDA levels of plasma and the severity of hemorrhages, congestion, edema, and leukocyte infiltration of bladder induced by CP were recovered with vitamin C and histidine treatments. Combined treatment with vitamin C and histidine showed a potentiation effect. The results indicated that vitamin C and histidine attenuated the CP-induced HC by reducing of free radical-induced toxic effects.

  9. Residuation theory

    CERN Document Server

    Blyth, T S; Sneddon, I N; Stark, M

    1972-01-01

    Residuation Theory aims to contribute to literature in the field of ordered algebraic structures, especially on the subject of residual mappings. The book is divided into three chapters. Chapter 1 focuses on ordered sets; directed sets; semilattices; lattices; and complete lattices. Chapter 2 tackles Baer rings; Baer semigroups; Foulis semigroups; residual mappings; the notion of involution; and Boolean algebras. Chapter 3 covers residuated groupoids and semigroups; group homomorphic and isotone homomorphic Boolean images of ordered semigroups; Dubreil-Jacotin and Brouwer semigroups; and loli

  10. An histidine covalent receptor/butenolide complex mediates strigolactone perception

    Science.gov (United States)

    Badet-Denisot, Marie-Ange; Pillot, Jean-Paul; Cornu, David; Le Caer, Jean-Pierre; Burger, Marco; Pelissier, Frank; Retailleau, Pascal; Turnbull, Colin; Bonhomme, Sandrine; Chory, Joanne; Rameau, Catherine; Boyer, François-Didier

    2016-01-01

    Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D-ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases and is known to hydrolyze the bond between the ABC lactone and the D-ring. Here we characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand. PMID:27479744

  11. In vitro study of proteolytic degradation of rat histidine decarboxylase.

    Science.gov (United States)

    Olmo, M T; Urdiales, J L; Pegg, A E; Medina, M A; Sánchez-Jiménez, F

    2000-03-01

    Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.

  12. L-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin

    Directory of Open Access Journals (Sweden)

    Schiffmann Elliott

    2005-02-01

    Full Text Available Abstract Background Autotaxin (ATX, NPP-2, originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD. The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity. Results We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion. Conclusion L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches.

  13. In silico study of fragile histidine triad interaction domains with MDM2 and p53

    OpenAIRE

    Ameneh Eslamparast; Mohammad Hossein Ghahremani; Soroush Sardari

    2014-01-01

    Background: Fragile histidine triad (FHIT) is considered as a member of the histidine triad (HIT) nucleotide-binding protein superfamily regarded as a putative tumor suppressor executing crucial role in inhibiting p53 degradation by MDM2. Accumulating evidences indicate FHIT interaction with p53 or MDM2; however, there is no certain study deciphering functional domains of FHIT involving in the interaction with MDM2 and/or p53. In this regard, such evident interaction can spring in mind determ...

  14. Proton transfer in histidine-tryptophan heterodimers embedded in helium droplets

    Energy Technology Data Exchange (ETDEWEB)

    Bellina, Bruno; Merthe, Daniel J.; Kresin, Vitaly V. [Department of Physics and Astronomy, University of Southern California, Los Angeles, California 90089-0484 (United States)

    2015-03-21

    We used cold helium droplets as nano-scale reactors to form and ionize, by electron bombardment and charge transfer, aromatic amino acid heterodimers of histidine with tryptophan, methyl-tryptophan, and indole. The molecular interaction occurring through an N–H ⋅ ⋅ ⋅ N hydrogen bond leads to a proton transfer from the indole group of tryptophan to the imidazole group of histidine in a radical cationic environment.

  15. Extracellular granzymes in inflammation

    NARCIS (Netherlands)

    Wensink, A.C.

    2014-01-01

    It has been well established that granzymes released by cytotoxic lymphocytes induce cell death in virus-infected cells and tumor cells. Next to this intracellular role of granzymes in triggering apoptosis, granzymes also exist extracellularly in the circulation of patients with autoimmune diseases

  16. A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis*

    Science.gov (United States)

    Williams, Sunanda Margrett; Chandran, Anu V.; Vijayabaskar, Mahalingam S.; Roy, Sourav; Balaram, Hemalatha; Vishveshwara, Saraswathi; Vijayan, Mamannamana; Chatterji, Dipankar

    2014-01-01

    Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8–2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release. PMID:24573673

  17. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J. (Virginia Tech); (UMC)

    2012-11-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

  18. The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima

    Energy Technology Data Exchange (ETDEWEB)

    Vu, Anh; Hamel, Damon J.; Zhou Hongjun; Dahlquist, Frederick W., E-mail: dahlquist@chem.ucsb.edu [University of California Santa Barbara, Department of Chemistry and Biochemistry (United States)

    2011-09-15

    The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought. In order to examine how the structures of Hpt domain homologs may differ from each other particularly in the conformation of the last helix, and whether an alternative conformation exists in the intact Hpt domain in solution, we have solved a high-resolution, solution structure of the CheA Hpt from T. maritima and characterized the backbone dynamics of this protein. The structure contains a four-helix bundle characteristic of histidine phosphotransfer domains. The position and orientation of the fifth helix resembles those in known Hpt domain crystal and solution structures in other histidine kinases. The alternative conformation that was reported in the crystal structure of the CheA Hpt from T. maritima missing the fifth helix is not detected in the solution structure, suggesting a role for the fifth helix in providing stabilizing forces to the overall structure.

  19. Tendon functional extracellular matrix.

    Science.gov (United States)

    Screen, Hazel R C; Berk, David E; Kadler, Karl E; Ramirez, Francesco; Young, Marian F

    2015-06-01

    This article is one of a series, summarizing views expressed at the Orthopaedic Research Society New Frontiers in Tendon Research Conference. This particular article reviews the three workshops held under the "Functional Extracellular Matrix" stream. The workshops focused on the roles of the tendon extracellular matrix, such as performing the mechanical functions of tendon, creating the local cell environment, and providing cellular cues. Tendon is a complex network of matrix and cells, and its biological functions are influenced by widely varying extrinsic and intrinsic factors such as age, nutrition, exercise levels, and biomechanics. Consequently, tendon adapts dynamically during development, aging, and injury. The workshop discussions identified research directions associated with understanding cell-matrix interactions to be of prime importance for developing novel strategies to target tendon healing or repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. ADI pathway and histidine decarboxylation are reciprocally regulated in Lactobacillus hilgardii ISE 5211: proteomic evidence.

    Science.gov (United States)

    Lamberti, Cristina; Purrotti, Micol; Mazzoli, Roberto; Fattori, Paolo; Barello, Cristina; Coïsson, Jean Daniel; Giunta, Carlo; Pessione, Enrica

    2011-07-01

    Amine production by amino acid decarboxylation is a common feature that is used by lactic acid bacteria (LAB) to complement lactic fermentation, since it is coupled with a proton-extruding antiport system which leads to both metabolic energy production and the attenuation of intracellular acidity. Analogous roles are played in LAB by both malolactic fermentation (MLF) and the arginine deiminase (ADI) pathway. The present investigation was aimed at establishing reciprocal interactions between amino acid decarboxylation and the two above mentioned routes. The analyses were carried out on a Lactobacillus hilgardii strain (ISE 5211) that is able to decarboxylate histidine to histamine, which had previously been isolated from wine and whose complete genome is still unknown. The 2DE proteomic approach, followed by MALDI TOF-TOF and De Novo Sequencing, was used to study the protein expression levels. The experimental evidence has indicated that malate does not influence histidine decarboxylase (HDC) biosynthesis and that histidine does not affect the malolactic enzyme level. However, the expression of the ADI route enzymes, arginine deiminase and ornithine transcarbamylase, is down-regulated by histidine: this biosynthetic repression is more important (4-fold) in cultures that are not supplemented with arginine, but is also significant (2-fold) in an arginine supplemented medium that normally induces the ADI pathway. On the other hand, arginine partially represses HDC expression, but only when histidine and arginine are both present in the culture medium. This proteomic study has also pointed out a down-regulation exerted by histidine over sugar metabolism enzymes and a GroEL stress protein. These data, together with the reciprocal antagonism between arginine deimination and histidine decarboxylation, offer clue keys to the understanding of the accumulation of lactate, amine, ammonia and ethylcarbamate in wine, with consequent implications on different health risk

  1. Residue processing

    Energy Technology Data Exchange (ETDEWEB)

    Gieg, W.; Rank, V.

    1942-10-15

    In the first stage of coal hydrogenation, the liquid phase, light and heavy oils were produced; the latter containing the nonliquefied parts of the coal, the coal ash, and the catalyst substances. It was the problem of residue processing to extract from these so-called let-down oils that which could be used as pasting oils for the coal. The object was to obtain a maximum oil extraction and a complete removal of the solids, because of the latter were returned to the process they would needlessly burden the reaction space. Separation of solids in residue processing could be accomplished by filtration, centrifugation, extraction, distillation, or low-temperature carbonization (L.T.C.). Filtration or centrifugation was most suitable since a maximum oil yield could be expected from it, since only a small portion of the let-down oil contained in the filtration or centrifugation residue had to be thermally treated. The most satisfactory centrifuge at this time was the Laval, which delivered liquid centrifuge residue and centrifuge oil continuously. By comparison, the semi-continuous centrifuges delivered plastic residues which were difficult to handle. Various apparatus such as the spiral screw kiln and the ball kiln were used for low-temperature carbonization of centrifuge residues. Both were based on the idea of carbonization in thin layers. Efforts were also being made to produce electrode carbon and briquette binder as by-products of the liquid coal phase.

  2. Production of extracellular amylase from agricultural residues by a ...

    African Journals Online (AJOL)

    ONOS

    2010-08-09

    Aug 9, 2010 ... Supplementation of carbon (starch) and nitrogen source (peptone) showed an increase in amylase production and the highest amount of amylase production obtained under all optimized conditions was 164 U/g. Key words: Solid state fermentation, optimization, Aspergillus, fermentation, amylases.

  3. Feeding filaggrin: effects of L-histidine supplementation in atopic dermatitis

    Directory of Open Access Journals (Sweden)

    Tan SP

    2017-10-01

    Full Text Available Siao Pei Tan,1,2 Simon B Brown,1,2 Christopher EM Griffiths,3 Richard B Weller,1,2 Neil K Gibbs3,4 1MRC Centre for Inflammation Research, 2Department of Dermatology, The University of Edinburgh, Edinburgh, 3Dermatology Centre, Division of Musculoskeletal and Dermatological Sciences, Salford Royal NHS Foundation Trust, University of Manchester, Manchester, 4Curapel, Life Sciences Hub Wales, Cardiff, UK Abstract: Atopic dermatitis (AD, also known as eczema, is one of the most common chronic skin conditions worldwide, affecting up to 16% of children and 10% of adults. It is incurable and has significant psychosocial and economic impacts on the affected individuals. AD etiology has been linked to deficiencies in the skin barrier protein, filaggrin. In mammalian skin, l-histidine is rapidly incorporated into filaggrin. Subsequent filaggrin proteolysis releases l-histidine as an important natural moisturizing factor (NMF. In vitro studies were conducted to investigate the influence of l-histidine on filaggrin processing and barrier function in human skin-equivalent models. Our further aim was to examine the effects of daily oral l-histidine supplementation on disease severity in adult AD patients. We conducted a randomized, double-blind, placebo-controlled, crossover, nutritional supplementation pilot study to explore the effects of oral l-histidine in adult AD patients (n=24. In vitro studies demonstrated that l-histidine significantly increased both filaggrin formation and skin barrier function (P<0.01, respectively. Data from the clinical study indicated that once daily oral l-histidine significantly reduced (P<0.003 AD disease severity by 34% (physician assessment using the SCORingAD tool and 39% (patient self-assessment using the Patient Oriented Eczema Measure tool after 4 weeks of treatment. No improvement was noted with the placebo (P>0.32. The clinical effect of oral l-histidine in AD was similar to that of mid-potency topical corticosteroids

  4. Conformationally Constrained Histidines in the Design of Peptidomimetics: Strategies for the χ-Space Control

    Directory of Open Access Journals (Sweden)

    Adriano Mollica

    2011-05-01

    Full Text Available A successful design of peptidomimetics must come to terms with χ-space control. The incorporation of χ-space constrained amino acids into bioactive peptides renders the χ1 and χ2 torsional angles of pharmacophore amino acids critical for activity and selectivity as with other relevant structural features of the template. This review describes histidine analogues characterized by replacement of native α and/or β-hydrogen atoms with alkyl substituents as well as analogues with α, β-didehydro unsaturation or Cα-Cβ cyclopropane insertion (ACC derivatives. Attention is also dedicated to the relevant field of β-aminoacid chemistry by describing the synthesis of β2- and β3-models (β-hHis. Structural modifications leading to cyclic imino derivatives such as spinacine, aza-histidine and analogues with shortening or elongation of the native side chain (nor-histidine and homo-histidine, respectively are also described. Examples of the use of the described analogues to replace native histidine in bioactive peptides are also given.

  5. Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

    Science.gov (United States)

    Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

  6. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

    Directory of Open Access Journals (Sweden)

    Zhen Cai

    2017-04-01

    Full Text Available As well as their importance to nutrition, fatty acids (FA represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK, RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp and catalytic ATP-binding (CA domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

  7. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

    Science.gov (United States)

    Cai, Zhen; Yuan, Zhi-Hui; Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li; Qian, Wei

    2017-04-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

  8. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence

    Science.gov (United States)

    Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li

    2017-01-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication. PMID:28369120

  9. Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, P.E.; Hartman, Z.; Citardi, M.J.

    1988-05-01

    Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H/sub 2/O/sub 2/ or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

  10. Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

    Science.gov (United States)

    Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Aréchiga-Figueroa, Iván A; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

    2017-03-01

    showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca(2+) ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10(-5.5) and 10(-9.0)  m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10(-5.5)  m, but 10(-9.0)  m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca(2+) ]i . © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  11. Nalpha-(1-deoxy-D-fructos-1-yl)-L-histidine ("D-Fructose-L-histidine"): a potent copper chelator from tomato powder.

    Science.gov (United States)

    Mossine, Valeri V; Mawhinney, Thomas P

    2007-12-12

    Dried fruits and vegetables are known for their high content of D-fructose-amino acids, or Amadori compounds, which appear at the initial step of the Maillard reaction and may participate in redox reactions mediated by trace metals. In this study, we investigated complexation between Cu(II) and N(alpha)-(1-deoxy-D-fructos-1-yl)-L-histidine (D-fructose-L-histidine, FruHis). The content of FruHis in two types of commercial tomato powders was estimated by GLC-MS, using single ion monitoring of trimethylsilylated FruHis hydroxyoximate, as 40 mg/100 g, whereas the concentration of free histidine in the powder samples was about 53 mg/100 g. The Cu(II)-binding ability of FruHis was studied along with structurally related molecules L-histidine, dipeptide L-carnosine, and N(alpha)-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg) at 25 degrees C using pH-potentiometric titrations. Analysis of the titration curves showed that formation of Cu(II)-FruHis complex species occurs at pH values as low as 2 and that the complexes were redox stable in the pH range 2-10.5, at least for the time of the experiment. At physiological pH, Cu(II) and FruHis form a dominant coordination species of composition MLH-1 (log beta = 5.67), with a presumably deprotonated anomeric hydroxyl group of the fructose portion. The apparent stability constant of 1:1 complexes formed by FruHis and Cu(II) in neutral aqueous solutions is about 10(4) times higher than similar values calculated for L-histidine, L-carnosine, and FruArg. FruHis nearly completely protected hydroxyl radical-mediated fragmentation of polymeric DNA in the presence of the Cu/H2O2/ascorbate system, whereas neither of the reference compounds could inhibit the DNA fragmentation as efficiently in similar conditions. These results warrant further investigation of FruHis as a potential food-related antioxidant.

  12. Synthesis and biology of ring-modified l-Histidine containing thyrotropin-releasing hormone (TRH) analogues.

    Science.gov (United States)

    Meena, Chhuttan L; Thakur, Avinash; Nandekar, Prajwal P; Sharma, Shyam S; Sangamwar, Abhay T; Jain, Rahul

    2016-03-23

    Thyrotropin-releasing hormone (TRH) analogues bearing halogen groups (Cl, Br and I) at the C-2 and/or C-5 position, and the alkyl group (CH3, C2H5, C3H7, CH2C6H5) at the N-1 position of the imidazole ring of the central histidine residue were synthesized and evaluated for the receptor binding, calcium mobilization (FLIPR), and IP-1 assay at the HEK mTRHR1 and HEK mTRHR2 expressing cell lines. The most promising analogue 7k showed 925-fold selectivity for HEK mTRH-R2 receptor subtype in the IP-1 assay, 272-fold selectivity for HEK mTRH-R2 receptor subtype in the FLIPR assay, and 21-fold receptor binding specificity at HEK TRH-R2 receptor subtype. The peptide 7k was evaluated in vitro in a brain membrane competitive binding assay, and for stability analysis in the presence of TRH-DE, in vivo. The analogue 7k showed decrease in the sleeping time by more than 76% in a pentobarbital-induced sleeping assay, and showed comparatively less elevation in the TSH level in the blood, in vivo. The computational homology modeling of TRH-R1 and TRH-R2 and docking study with the most potent peptide 7k provide impetus to design CNS specific TRH analogues. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Activity of two histidine decarboxylases from Photobacterium phosphoreum at different temperatures, pHs, and NaCl concentrations.

    Science.gov (United States)

    Morii, Hideaki; Kasama, Kentaro

    2004-08-01

    The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible enzymes. This article reports the effect of various growth and reaction conditions, such as temperature, pH, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P. phosphoreum. The histidine decarboxylase activity of the cell-free extracts was highest in 7 degrees C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at pH 6.0. Moreover, percent activity of the constitutive and inducible enzymes was highest for the inducible enzyme in cultures grown at 7 degrees C and pH 7.5 and in 5% NaCl. The temperature and pH dependences of histidine decarboxylase differed between the constitutive and inducible enzymes; that is, the activity of histidine decarboxylases was optimum at 30 degrees C and pH 6.5 for the inducible enzyme and 40 degrees C and pH 6.0 for the constitutive enzyme. The differences in the temperature and pH dependences between the two enzymes extended the activity range of histidine decarboxylase under reaction conditions. On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two enzymes. Additionally, the effects of reaction temperature, pH, and NaCl concentration on the constitutive enzyme activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive enzyme activity reflected the whole histidine decarboxylase activity.

  14. Histidine-mediated xylem loading of zinc is a species-wide character in Noccaea caerulescens.

    NARCIS (Netherlands)

    Kozhevnikova, A.; Seregin, I.V.; Erlikh, N.T.; Shevyreva, T.A.; Andreev, I.M.; Verweij, R.; Schat, H.

    2014-01-01

    Histidine plays a crucial role in nickel (Ni) translocation in Ni-hyperaccumulating plants. Here, we investigated its role in zinc (Zn) translocation in four accessions of the Zn hyperaccumulator, Noccaea caerulescens, using the related non-hyperaccumulator, Thlaspi arvense, as a reference. We

  15. Molecular cloning and differential IgG responses to a histidine-rich ...

    African Journals Online (AJOL)

    In order to further investigate host-parasite interactions in onchocerciasis, a major Onchocerca volvulus histidine rich antigen termed OvL3.C1 was isolated from an O. volvulus cDNA library using antibodies from putatively immune subjects living in onchocerciasis endemic communities in Cameroon. Analysis of its ...

  16. Histidine as a catalyst in organic synthesis: A facile in situ synthesis ...

    Indian Academy of Sciences (India)

    Unknown

    (Aldrich, E-Merck and Acros) and were purified prior to use either by distillation or by recrystallization. Histidine (E-Merck) ... ethylacetate (E-Merck) were purified by distillation before use. Double distilled water, .... Sandler S R and Karo W 1972 Organic functional group preparations (New York: Academic. Press) vol. 3. 4.

  17. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays

    DEFF Research Database (Denmark)

    Henriksen, Signe Teuber; Liu, J.; Estiu, G.

    2010-01-01

    in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking...

  18. β-Alanyl-L-Histidine, an Anti-Oxidant, Anti-fibrotic and Geno ...

    African Journals Online (AJOL)

    ... hepatic hydroxyproline, protein carbonyl, hydrogen peroxide (H2O2) levels, DNA damage, Cytochrome P4502E1 (CYP2E1) activity and transforming growth factor-β1 (TGF-β1) mRNA level. In conclusion: β-alanyl-L-histidine possesses hepatoprotective properties through reducing hepatic toxicity markers, oxidative stress, ...

  19. Histidine as a catalyst in organic synthesis: A facile in situ synthesis ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 113; Issue 4. Histidine as a catalyst in organic synthesis: A facile in situ synthesis of , N-diarylnitrones. H Mallesha K R Ravi Kumar B K Vishu Kumar K Mantelingu K S Rangappa. Organic Volume 113 Issue 4 August 2001 pp 291-296 ...

  20. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

    Science.gov (United States)

    Hernández-Sánchez, Itzell E.; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P.; Jiménez-Bremont, Juan F.

    2015-01-01

    The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization. PMID:26442018

  1. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

    Directory of Open Access Journals (Sweden)

    Itzell Euridice Hernández-Sánchez

    2015-09-01

    Full Text Available The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

  2. Gas-phase structures and thermochemistry of neutral histidine and its conjugated acid and base.

    Science.gov (United States)

    Riffet, Vanessa; Bouchoux, Guy

    2013-04-28

    Extensive exploration of the conformational space of neutral, protonated and deprotonated histidine has been conducted at the G4MP2 level. Theoretical protonation and deprotonation thermochemistry as well as heats of formation of gaseous histidine and its ionized forms have been calculated at the G4 level considering either the most stable conformers or an equilibrium population of conformers at 298 K. These theoretical results were compared to evaluated experimental determinations. Recommended proton affinity and protonation entropy deduced from these comparisons are PA(His) = 980 kJ mol(-1) and ΔpS(His) ∼ 0 J mol(-1) K(-1), thus leading to a gas-phase basicity value of GB(His) = 947.5 kJ mol(-1). Similarly, gas phase acidity parameters are ΔacidH(o)(His) = 1373 kJ mol(-1), ΔacidS(His) ∼ 10 J mol(-1) K(-1) and ΔacidG(o)(His) = 1343 kJ mol(-1). Computed G4 heats of formation values are equal to -290, 265 and -451 kJ mol(-1) for gaseous neutral histidine and its protonated and deprotonated forms, respectively. The present computational data correct, and complete, previous thermochemical parameter estimates proposed for gas-phase histidine and its acido-basic properties.

  3. Highly Efficient Photocatalytic Hydrogen Production of Flower-like Cadmium Sulfide Decorated by Histidine

    National Research Council Canada - National Science Library

    Wang, Qizhao; Lian, Juhong; Li, Jiajia; Wang, Rongfang; Huang, Haohao; Su, Bitao; Lei, Ziqiang

    2015-01-01

    ...•4H2O and thiourea as precursors and L-Histidine as a chelating agent. The morphology, crystal phase, and photoelectrochemical performance of the flower-like CdS and pure CdS nanocrystals are carefully investigated via various characterizations...

  4. Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease

    NARCIS (Netherlands)

    Bartneck, M.; Fech, V.; Ehling, J.; Govaere, O.; Warzecha, K.T.; Hittatiya, K.; Vucur, M.; Gautheron, J.; Luedde, T.; Trautwein, C.; Lammers, Twan Gerardus Gertudis Maria; Roskams, T.; Jahnen-Dechent, W.; Tacke, F.

    2016-01-01

    Pathogen- and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to

  5. C@Fe 3 O 4 /NTA-Ni magnetic nanospheres purify histidine-tagged ...

    African Journals Online (AJOL)

    This study reports synthesis of Ni-nitrilotriacetic acid (Ni-NTA) modified carbon nanospheres containing magnetic Fe3O4 particles (C@Fe3O4), which can act as a general tool to separate and purify histidine-tagged fetidin. In this experiment, C nanospheres are prepared from glucose using the hydrothermal process, ...

  6. Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

    Science.gov (United States)

    Anderson, P M; Korte, J J; Holcomb, T A

    1994-11-29

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Muscle histidine-containing dipeptides are elevated by glucose intolerance in both rodents and men.

    Directory of Open Access Journals (Sweden)

    Sanne Stegen

    Full Text Available Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats, 60% high-fat feeding (male rats, cafeteria feeding (male rats, 70% high-fat feeding (female mice. Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients and muscle carnosine and gene expression of muscle fiber type markers were measured.Diet interventions in rodents (cafeteria and 70% high-fat feeding induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1, +30% (p<0.05 and +39% (p<0.05, respectively. The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05 and tended to increase in the diabetic men (1.6-fold, p = 0.07, compared to healthy lean subjects.Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional. In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional. Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.

  8. Preeclampsia and Extracellular Vesicles.

    Science.gov (United States)

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

  9. RNA in extracellular vesicles.

    Science.gov (United States)

    Kim, Kyoung Mi; Abdelmohsen, Kotb; Mustapic, Maja; Kapogiannis, Dimitrios; Gorospe, Myriam

    2017-07-01

    Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40-1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413 For further resources related to this article, please visit the WIREs website. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  10. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Marc Keppel

    2018-02-01

    Full Text Available The HrrSA and the ChrSA two-component systems play a central role in the coordination of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum and the prominent pathogen Corynebacterium diphtheriae, both members of the Corynebacteriaceae. In this study, we have performed a comparative analysis of the membrane topology and heme-binding characteristics of the histidine kinases HrrS and ChrS of C. glutamicum. While the cytoplasmic catalytic domains are highly conserved between HrrS and ChrS, the N-terminal sensing parts share only minor sequence similarity. PhoA and LacZ fusions of the N-terminal sensor domains of HrrS and ChrS revealed that both proteins are embedded into the cytoplasmic membrane via six α-helices. Although the overall membrane topology appeared to be conserved, target gene profiling indicated a higher sensitivity of the ChrS system to low heme levels (< 1 μM. In vitro, solubilized and purified full-length proteins bound heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain revealed three aromatic residues (Y112, F115, and F118, which apparently contribute to heme binding of HrrS. Exchange of either one or all three residues resulted in an almost abolished heme binding of HrrS in vitro. In contrast, ChrS mutants only displayed a red shift of the soret band from 406 to 418 nm suggesting an altered set of ligands in the triple mutant. In line with target gene profiling, these in vitro studies suggest distinct differences in the heme-protein interface of HrrS and ChrS. Since the membrane topology mapping displayed no extensive loop regions and alanine-scanning revealed potential heme-binding residues in α-helix number four, we propose an intramembrane sensing mechanism for both proteins. Overall, we present a first comparative analysis of the ChrS and HrrS kinases functioning as transient heme sensors in the Corynebacteriaceae.

  12. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable...... to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  13. Neutrophil Extracellular Traps, Antiphospholipid Antibodies and Treatment

    National Research Council Canada - National Science Library

    Jessica Bravo-Barrera; Maria Kourilovitch; Claudio Galarza-Maldonado

    2017-01-01

    Neutrophil extracellular traps (NETs) are a network of extracellular fibers, compounds of chromatin, neutrophil DNA and histones, which are covered with antimicrobial enzymes with granular components...

  14. Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein▿

    OpenAIRE

    Qin, Zhao-ling; Zheng, Yan; Kielian, Margaret

    2009-01-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reac...

  15. Extracellular vesicles in renal disease.

    Science.gov (United States)

    Karpman, Diana; Ståhl, Anne-Lie; Arvidsson, Ida

    2017-09-01

    Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

  16. Inactivation of histidine decarboxylase by gamma irradiation for controlling histamine formation

    Science.gov (United States)

    Pak, Won-Min; Kim, Koth-Bong-Woo-Ri; Kim, Min-Ji; Ahn, Dong-Hyun

    2017-12-01

    In this study, the effects of gamma irradiation on the survival of Morganella morganii and Photobacterium phosphoreum and the activity of their crude histidine decarboxylase (HDC) were investigated. The two strains and their crude HDC were irradiated up to 10 kGy. Viable cells of M. morganii and P. phosphoreum were not detected at any dose. The activity of crude HDC was decreased with increasing dose. In particular, the gamma irradiation at 5 and 10 kGy resulted in > 90% inactivation of crude HDC from M. morganii and P. phosphoreum, respectively. In SDS-PAGE and native PAGE, slight structural changes of crude HDC appeared with gamma irradiation. These results suggest that gamma irradiation is effective in reducing histamine production through inactivation survival of M. morganii and P. phosphoreum, and their histidine decarboxylase activity.

  17. Growth and characterization of an organic nonlinear optical material: L-Histidine malonate

    Science.gov (United States)

    Ramya, K.; Saraswathi, N. T.; Raja, C. Ramachandra

    2016-10-01

    L-Histidine malonate is one of the potential organic material for nonlinear optical applications. Single crystals of L-Histidine malonate were grown by the liquid diffusion method. The lattice parameter values were evaluated from single crystal X-ray diffraction technique. The Fourier Transform Infra Red and Raman spectral studies were employed to identify the different modes of vibrations of molecular groups in the crystal. Optical characterization and the percentage of optical transmission were recorded using UV-vis-NIR spectroscopy. The molecular structure was established by proton and carbon Nuclear magnetic resonance spectral studies. The thermal behavior of the material has been studied by Thermo gravimetric and Differential thermal plots. The second harmonic generation conversion efficiency was found out from the powder technique of Kurtz and Perry.

  18. Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Rung-Yi; Huang, Siyu; Fenwick, Michael K.; Hazra, Amrita; Zhang, Yang; Rajashankar, Kanagalaghatta; Philmus, Benjamin; Kinsland, Cynthia; Sanders, Jennie Mansell; Ealick, Steven E.; Begley, Tadhg P. (Cornell); (TAM)

    2012-06-26

    In Saccharomyces cerevisiae, thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.

  19. Effects of phorbol ester and dexamethasone treatment on histidine decarboxylase and ornithine decarboxylase in basophilic cells.

    Science.gov (United States)

    Fajardo, I; Urdiales, J L; Medina, M A; Sanchez-Jimenez, F

    2001-05-01

    Both histamine and polyamines are important for maintaining basophilic cell function and viability. The synthesis of these biogenic amines is regulated by histidine decarboxylase and ornithine decarboxylase, respectively. In other mammalian tissues, an interplay between histamine and polyamine metabolisms has been suspected. In this report, the interplay between histamine and ornithine-derived polyamines was studied in a non-transformed mouse mast cell line (C57.1) treated with phorbol ester and dexamethasone, a treatment previously used to increase histidine decarboxylase expression in mastocytoma and basophilic leukemia. Treatment with phorbol ester and dexamethasone increased histidine decarboxylase expression and intracellular histamine levels in C57.1 mast cells to a greater extent than those found for other transformed basophilic models. The treatment also induced a reduction in ornithine decarboxylase expression, intracellular polyamine contents, and cell proliferation. These results indicate that the treatment induces a co-ordinate response of polyamine metabolism and proliferation in mast cells and other immune-related cells. The decrease in the proliferative capacity of mast cells caused by phorbol ester and dexamethasone was simultaneous to an increase in histamine production. Our results, together with those reported by other groups working with polyamine-treated mast cells, indicate an antagonism between histamine and polyamines in basophilic cells.

  20. Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk.

    Science.gov (United States)

    Kurut, Anıl; Henriques, João; Forsman, Jan; Skepö, Marie; Lund, Mikael

    2014-04-01

    Histidine-rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are-in contrast to the majority of proteins-optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)-histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine-rich antimicrobial peptides. Copyright © 2013 Wiley Periodicals, Inc.

  1. Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study

    Directory of Open Access Journals (Sweden)

    Yan-Chuan Li

    2016-07-01

    Full Text Available Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC. Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p < 0.05 and obese (p < 0.01 participants of both sexes. Dietary histidine was inversely associated with body mass index (BMI, waist circumference (WC and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG, homeostasis model assessment of insulin resistance (HOMA-IR, 2-h postprandial glucose (2 h-PG, tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, interleukin-6 (IL-6, C-reactive protein (CRP, malonaldehyde (MDA and vaspin and higher glutathione peroxidase (GSH-Px, superoxide dismutase (SOD and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults.

  2. Structural-dynamical investigation of the ZnuA histidine-rich loop: involvement in zinc management and transport.

    Science.gov (United States)

    Falconi, Mattia; Oteri, Francesco; Di Palma, Francesco; Pandey, Saurabh; Battistoni, Andrea; Desideri, Alessandro

    2011-02-01

    Comparative homology modelling techniques have been used to model the protein ZnuA from Salmonella enterica serovar Typhimurium using the 3D structure of the homologous protein from Escherichia coli. These two-domain proteins bind one Zn(2+) atom, with high affinity, in the inter-domain cleft and possess a histidine-rich loop in the N-terminal domain. Alternative structures of the ZnuA histidine-rich loop, never resolved by the X-ray diffraction method, have been modelled. A model of the apo form, one with the histidine-rich loop deleted and two alternative structures with a second zinc ion bound to the histidine-rich loop, have been generated. In all the modelled proteins, investigated through molecular dynamics simulation, the histidine-rich loop is highly mobile and its fluctuations are correlated to the ligand stability observed in the zinc sites. Based on the plasticity of the histidine-rich loop and its significant effects on protein mobility a possible role in the capture and/or transfer of the zinc ions has been suggested.

  3. N-Acetylglucosaminidases from CAZy Family GH3 Are Really Glycoside Phosphorylases, Thereby Explaining Their Use of Histidine as an Acid/Base Catalyst in Place of Glutamic Acid*

    Science.gov (United States)

    Macdonald, Spencer S.; Blaukopf, Markus; Withers, Stephen G.

    2015-01-01

    CAZy glycoside hydrolase family GH3 consists primarily of stereochemistry-retaining β-glucosidases but also contains a subfamily of β-N-acetylglucosaminidases. Enzymes from this subfamily were recently shown to use a histidine residue within a His-Asp dyad contained in a signature sequence as their catalytic acid/base residue. Reasons for their use of His rather than the Glu or Asp found in other glycosidases were not apparent. Through studies on a representative member, the Nag3 β-N-acetylglucosaminidase from Cellulomonas fimi, we now show that these enzymes act preferentially as glycoside phosphorylases. Their need to accommodate an anionic nucleophile within the enzyme active site explains why histidine is used as an acid/base catalyst in place of the anionic glutamate seen in other GH3 family members. Kinetic and mechanistic studies reveal that these enzymes also employ a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate, which was directly detected by mass spectrometry. Phosphate has no effect on the rates of formation of the glycosyl-enzyme intermediate, but it accelerates turnover of the N-acetylglucosaminyl-enzyme intermediate ∼3-fold, while accelerating turnover of the glucosyl-enzyme intermediate several hundredfold. These represent the first reported examples of retaining β-glycoside phosphorylases, and the first instance of free β-GlcNAc-1-phosphate in a biological context. PMID:25533455

  4. An analysis of solution structure and signaling mechanism of LovK, a sensor histidine kinase integrating light and redox signals†

    Science.gov (United States)

    Purcell, Erin B.; McDonald, Claudia A.; Palfey, Bruce A.; Crosson, Sean

    2010-01-01

    Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria. These ≈100 residue photosensory modules are generally encoded within larger, multi-domain proteins that control a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes a soluble LOV-histidine kinase, LovK, that regulates the adhesive properties of the cell. Full-length LovK is dimeric as are a series of systematically truncated LovK constructs containing only the N-terminal LOV sensory domain. Non-conserved sequence flanking the LOV domain functions to tune the signaling lifetime of the protein. Size exclusion chromatography and small angle X-ray scattering (SAXS) demonstrate that the LOV sensor domain does not undergo a large conformational change in response to photon absorption. However, limited proteolysis identifies a sequence flanking the C-terminus of the LOV domain as a site of light-induced change in protein conformation/dynamics. Based on SAXS envelope reconstruction and bioinformatic prediction, we propose this dynamic region of structure is an extended C-terminal coiled-coil that links the LOV domain to the histidine kinase domain. To test the hypothesis that LOV domain signaling is affected by cellular redox state in addition to light, we measured the reduction potential of the LovK FMN cofactor. The measured potential of −258 mV is congruent with the redox potential of gram-negative cytoplasm during logarithmic growth (−260 to −280 mV). Thus a fraction of LovK in the cytosol may be in the reduced state under typical growth conditions. Chemical reduction of the FMN cofactor of LovK attenuates light-dependent ATPase activity of the protein in vitro, demonstrating that LovK can function as a conditional photosensor that is regulated by the oxidative state of the cellular environment. PMID:20593779

  5. Extracellular Vesicles in Renal Pathophysiology.

    Science.gov (United States)

    Pomatto, Margherita A C; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

  6. Extracellular matrix and wound healing.

    Science.gov (United States)

    Maquart, F X; Monboisse, J C

    2014-04-01

    Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Combination treatment for allergic conjunctivitis - Plant derived histidine decarboxylase inhibitor and H1 antihistaminic drug.

    Science.gov (United States)

    Bakrania, Anita K; Patel, Snehal S

    2015-08-01

    Aim of present investigation was to study the effect of catechin and the combination of catechin and cetirizine in ovalbumin induced animal model of allergic conjunctivitis. Guinea pigs were divided into 5 groups: normal control, disease control, disease treated with catechin 100 mg/kg, disease treated with cetirizine 10 mg/kg, disease treated with combination of catechin and cetirizine, 50 mg/kg & 5 mg/kg respectively. Sensitization was carried out by intraperitoneal injection of ovalbumin for the period of 14 day. Simultaneously, catechin was administered orally for 14 days while, cetirizine was administered at the day of experiment. Determination of clinical scoring, mast cell and blood histamine content, histidine decarboxylase activity from stomach was carried out. Vascular permeability was measured by dye leakage after secondary challenge of allergen and conjunctival tissues were subjected for histopathological examinations. Treatment with catechin, cetirizine and combination showed significant (P < 0.05) decrease in clinical scoring and vascular permeability. While, catechin 100 mg/kg and catechin 50 mg/kg showed significant (P < 0.05) decrease in histamine content in mast and blood. The treatment also showed significant (P < 0.05) decrease in the histidine decarboxylase enzyme activity. However, cetirizine group did not show any difference in enzyme activity as well as histamine content. Histopathological examination also showed improvement in ulceration and decrease in edema and inflammation in all treatment groups. From the present study, we can conclude that catechin exhibits potent anti-allergic activity by histidine decarboxylase enzyme inhibition and combination shown significant anti-allergic activity at reduced dose by both enzyme inhibition as well as inhibition of histamine receptors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik

    2012-01-01

    In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without...... a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole...

  9. Insufficient intake of L-histidine reduces brain histamine and causes anxiety-like behaviors in male mice.

    Science.gov (United States)

    Yoshikawa, Takeo; Nakamura, Tadaho; Shibakusa, Tetsuro; Sugita, Mayu; Naganuma, Fumito; Iida, Tomomitsu; Miura, Yamato; Mohsen, Attayeb; Harada, Ryuichi; Yanai, Kazuhiko

    2014-10-01

    L-histidine is one of the essential amino acids for humans, and it plays a critical role as a component of proteins. L-histidine is also important as a precursor of histamine. Brain histamine is synthesized from L-histidine in the presence of histidine decarboxylase, which is expressed in histamine neurons. In the present study, we aimed to elucidate the importance of dietary L-histidine as a precursor of brain histamine and the histaminergic nervous system. C57BL/6J male mice at 8 wk of age were assigned to 2 different diets for at least 2 wk: the control (Con) diet (5.08 g L-histidine/kg diet) or the low L-histidine diet (LHD) (1.28 g L-histidine/kg diet). We measured the histamine concentration in the brain areas of Con diet-fed mice (Con group) and LHD-fed mice (LHD group). The histamine concentration was significantly lower in the LHD group [Con group vs. LHD group: histamine in cortex (means ± SEs): 13.9 ± 1.25 vs. 9.36 ± 0.549 ng/g tissue; P = 0.002]. Our in vivo microdialysis assays revealed that histamine release stimulated by high K(+) from the hypothalamus in the LHD group was 60% of that in the Con group (P = 0.012). However, the concentrations of other monoamines and their metabolites were not changed by the LHD. The open-field tests showed that the LHD group spent a shorter amount of time in the central zone (87.6 ± 14.1 vs. 50.0 ± 6.03 s/10 min; P = 0.019), and the light/dark box tests demonstrated that the LHD group spent a shorter amount of time in the light box (198 ± 8.19 vs. 162 ± 14.1 s/10 min; P = 0.048), suggesting that the LHD induced anxiety-like behaviors. However, locomotor activity, memory functions, and social interaction did not differ between the 2 groups. The results of the present study demonstrated that insufficient intake of histidine reduced the brain histamine content, leading to anxiety-like behaviors in the mice. © 2014 American Society for Nutrition.

  10. A modified suspension test for estimating the mutagenicity of samples containing free and (or) protein-bound histidine.

    Science.gov (United States)

    Liu, Bo; Jin, Jianling; Cheng, Yanfei; Zhang, Huaiqiang; Gao, Peiji

    2009-02-01

    The Ames test has not been very effective in estimating the mutagenicity of histidine-containing samples because external free and (or) protein-bound histidine in these samples would allow the histidine auxotrophs in such test samples to grow more compared with the negative controls that were used as the reference. This could give rise to a false positive.n this study, a modified suspension mutagenicity assay (MS assay) was developed. The tester strains were incubated in Luria-Bertani (LB) broth containing different concentrations of traditional Chinese medicines (TCMs) until the declining phase, and the test samples were assayed to be mutagenic or not by observing whether statistically significant differences were demonstrated in the relative reversion frequencies (RRFs) between the negative control groups and the test groups. Collectively, using LB broth as the test medium and comparing the RRFs in the declining phase made this assay less influenced by the presence of histidine in the test samples.The mutagenicity of some TCMs was measured with the MS assay. The results in MS assay were consistent with those in the mammalian bone marrow chromosomal aberration test, which indicated that the MS assay was appropriate to estimate the mutagenicity of samples containing free and (or) protein-bound histidine.

  11. Glomerular extracellular matrix components and integrins

    NARCIS (Netherlands)

    Sterk, L. M.; de Melker, A. A.; Kramer, D.; Kuikman, I.; Chand, A.; Claessen, N.; Weening, J. J.; Sonnenberg, A.

    1998-01-01

    It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular

  12. Copper radical oxidases and related extracellular oxidoreductases of wood-decay Agaricomycetes

    Science.gov (United States)

    Phil Kersten; Dan Cullen

    2014-01-01

    Extracellular peroxide generation, a key component of oxidative lignocellulose degradation, has been attributed to various enzymes including the copper radical oxidases. Encoded by a family of structurally related sequences, the genes are widely distributed among wood decay fungi including three recently completed polypore genomes. In all cases, core catalytic residues...

  13. Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri.

    Science.gov (United States)

    Wenzl, S; Thym, D; Sumper, M

    1984-04-01

    We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 112). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix.

  14. Extracellular vesicles in cardiovascular homeostasis and disease.

    Science.gov (United States)

    Hutcheson, Joshua D; Aikawa, Elena

    2018-02-19

    Extracellular vesicles have emerged as one of the most important means through which cells interact with each other and the extracellular environment, but extracellular vesicle research remains challenging due to their small size, limited amount of material required for traditional molecular biology assays and inconsistency in the methods of their isolation. The advent of new technologies and standards in the field, however, have led to increased mechanistic insight into extracellular vesicle function. Herein, the latest studies on the role of extracellular vesicles in cardiovascular physiology and disease are discussed. Extracellular vesicles help control cardiovascular homeostasis and remodelling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment. The message carried from the parent cell to extracellular space can be intended for both local (within the same tissue) and distal (downstream of blood flow) targets. Pathological cargo loaded within extracellular vesicles could further result in various diseases. On the contrary, new studies indicate that injection of extracellular vesicles obtained from cultured cells into diseased tissues can promote restoration of normal tissue function. Extracellular vesicles are an integral part of cell and tissue function, and harnessing the properties inherent to extracellular vesicles may provide a therapeutic strategy to promote tissue regeneration.

  15. Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins

    Science.gov (United States)

    Aygar, Gülfem; Kaya, Murat; Özkan, Necati; Kocabıyık, Semra; Volkan, Mürvet

    2015-12-01

    Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (-COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2-CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.

  16. Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury.

    Science.gov (United States)

    Kanki, Masashi; Yoda, Tomoko; Tsukamoto, Teizo; Baba, Eiichiroh

    2007-03-01

    Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

  17. Histidine Decarboxylases and Their Role in Accumulation of Histamine in Tuna and Dried Saury▿

    Science.gov (United States)

    Kanki, Masashi; Yoda, Tomoko; Tsukamoto, Teizo; Baba, Eiichiroh

    2007-01-01

    Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae. PMID:17220267

  18. Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes.

    Science.gov (United States)

    Lächelt, Ulrich; Kos, Petra; Mickler, Frauke M; Herrmann, Annika; Salcher, Eveline E; Rödl, Wolfgang; Badgujar, Naresh; Bräuchle, Christoph; Wagner, Ernst

    2014-01-01

    The cationizable nature of 'proton-sponge' transfection agents facilitates pDNA delivery in several steps. Protonated amines account for electrostatic DNA binding and cellular uptake, buffering amines mediate polyplex escape from acidifying intracellular vesicles. As demonstrated with a sequence-defined library of oligo(ethanamino)amides containing selected oligoethanamino acids and histidines, the total protonation capacity as well as the cationization pH profile within the endolysosomal range have critical impact on gene transfer. Building blocks with even numbered amine groups (Gtt, Sph) exhibited higher total endolysosomal buffer capacity than odd number (Stp) analogs. Within the endolysosomal range, Gtt has the highest buffer capacity around pH5, whereas Stp has its maximum around pH7. Histidines increased the total buffer capacity, resulted in a more continuous cationization pH profile and greatly improved transgene expression in vitro and in vivo. Using receptor targeted and polyethylene glycol shielded polyplexes, better endosomal escape and >100-fold enhanced transfection was detected. Proton-sponge transfection agents for pDNA delivery are characterized in this study, demonstrating over 100-fold enhanced transection and better endosomal escape by using receptor targeted and polyethylene glycol shielded polyplexes. © 2013.

  19. Novel Organotin(IV) Schiff Base Complexes with Histidine Derivatives: Synthesis, Characterization, and Biological Activity

    Science.gov (United States)

    Garza-Ortiz, Ariadna; Camacho-Camacho, Carlos; Sainz-Espuñes, Teresita; Rojas-Oviedo, Irma; Gutiérrez-Lucas, Luis Raúl; Gutierrez Carrillo, Atilano; Vera Ramirez, Marco A.

    2013-01-01

    Five novel tin Schiff base complexes with histidine analogues (derived from the condensation reaction between L-histidine and 3,5-di-tert-butyl-2-hydroxybenzaldehyde) have been synthesized and characterized. Characterization has been completed by IR and high-resolution mass spectroscopy, 1D and 2D solution NMR (1H, 13C  and 119Sn), as well as solid state 119Sn NMR. The spectroscopic evidence shows two types of structures: a trigonal bipyramidal stereochemistry with the tin atom coordinated to five donating atoms (two oxygen atoms, one nitrogen atom, and two carbon atoms belonging to the alkyl moieties), where one molecule of ligand is coordinated in a three dentate fashion. The second structure is spectroscopically described as a tetrahedral tin complex with four donating atoms (one oxygen atom coordinated to the metal and three carbon atoms belonging to the alkyl or aryl substituents), with one molecule of ligand attached. The antimicrobial activity of the tin compounds has been tested against the growth of bacteria in vitro to assess their bactericidal properties. While pentacoordinated compounds 1, 2, and 3 are described as moderate effective to noneffective drugs against both Gram-positive and Gram-negative bacteria, tetracoordinated tin(IV) compounds 4 and 5 are considered as moderate effective and most effective compounds, respectively, against the methicillin-resistant Staphylococcus aureus strains (Gram-positive). PMID:23864839

  20. Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial and fungal infection.

    Science.gov (United States)

    Pham, Jasmine; Liu, Jasmine; Bennett, Mark H; Mansfield, John W; Desikan, Radhika

    2012-04-01

    • The ability of plants to adapt to multiple stresses imposed by the natural environment requires cross-talk and fine-tuning of stress signalling pathways. The hybrid histidine kinase Arabidopsis histidine kinase 5 (AHK5) is known to mediate stomatal responses to exogenous and endogenous signals in Arabidopsis thaliana. The purpose of this study was to determine whether the function of AHK5 in stress signalling extends beyond stomatal responses. • Plant growth responses to abiotic stresses, tissue susceptibility to bacterial and fungal pathogens, and hormone production and metabolism of reactive oxygen species were monitored in a T-DNA insertion mutant of AHK5. • The findings of this study indicate that AHK5 positively regulates salt sensitivity and contributes to resistance to the bacterium Pseudomonas syringae pv. tomato DC3000 and the fungal pathogen Botrytis cinerea. • This is the first report of a role for AHK5 in the regulation of survival following challenge by a hemi-biotrophic bacterium and a necrotrophic fungus, as well as in the growth response to salt stress. The function of AHK5 in regulating the production of hormones and redox homeostasis is discussed. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  1. Selective histamine uptake rescues photo- and mechanoreceptor function of histidine decarboxylase-deficient Drosophila mutant.

    Science.gov (United States)

    Melzig, J; Burg, M; Gruhn, M; Pak, W L; Buchner, E

    1998-09-15

    In insects, histamine is found both in the peripheral nervous system (PNS) and in the CNS and is known to function as a fast neurotransmitter in photoreceptors that have been shown to express selectively the hdc gene. This gene codes for histidine decarboxylase (HDC), the enzyme for histamine synthesis. Fast neurotransmission requires the efficient removal of the transmitter from the synaptic cleft. Here we identify in Drosophila photo- and mechanoreceptors a histamine uptake mechanism that can restore the function of these receptors in mutants unable to synthesize histamine. When apparent null mutants for the hdc gene imbibe aqueous histamine solution or are genetically "rescued" by a transgene ubiquitously expressing histidine decarboxylase under heat-shock control, sufficient amounts of histamine selectively accumulate in photo- and mechanoreceptors to generate near-normal electrical responses in second-order visual interneurons and qualitatively to restore wild-type visual and mechanosensory behavior. This strongly supports the proposal that histamine functions as a fast neurotransmitter also in a certain class of mechanoreceptors. A set of CNS-intrinsic neurons that in the wild type contain high concentrations of histamine apparently lacks this uptake mechanism. We therefore speculate that histamine of intrinsic neurons may function as a neuromodulator rather than as a fast transmitter.

  2. Extracellular Vesicles in Cardiovascular Theranostics

    OpenAIRE

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S.; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro,Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells pla...

  3. Immunotherapeutic Potential of Extracellular Vesicles

    OpenAIRE

    Zhang, Bin; Yin, Yijun; Lai, Ruenn Chai; Lim, Sai Kiang

    2014-01-01

    Extracellular vesicle or EV is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes, the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized...

  4. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  5. Extracellular genomic biomarkers of osteoarthritis.

    Science.gov (United States)

    Budd, Emma; Nalesso, Giovanna; Mobasheri, Ali

    2018-01-01

    Osteoarthritis (OA), a chronic, debilitating and degenerative disease of the joints, is the most common form of arthritis. The seriousness of this prevalent and chronic disease is often overlooked. Disease modifying OA drug development is hindered by the lack of soluble biomarkers to detect OA early. The objective of OA biomarker research is to identify early OA prior to the appearance of radiographic signs and the development of pain. Areas covered: This review has focused on extracellular genomic material that could serve as biomarkers of OA. Recent studies have examined the expression of extracellular genomic material such as miRNA, lncRNA, snoRNA, mRNA and cell-free DNA, which are aberrantly expressed in the body fluids of OA patients. Changes in genomic content of peripheral blood mononuclear cells in OA could also function as biomarkers of OA. Expert commentary: There is an unmet need for soluble biomarkers for detecting and then monitoring OA disease progression. Extracellular genomic material research may also reveal more about the underlying pathophysiology of OA. Minimally-invasive liquid biopsies such as synovial fluid and blood sampling of genomic material may be more sensitive over radiography in the detection, diagnosis and monitoring of OA in the future.

  6. Extracellular Vesicles in Lung Disease.

    Science.gov (United States)

    Kubo, Hiroshi

    2018-01-01

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  7. Chemical conversion of cisplatin and carboplatin with histidine in a model protein crystallized under sodium iodide conditions

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M.; Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    Crystals of HEWL with cisplatin and HEWL with carboplatin grown in sodium iodide conditions both show a partial chemical transformation of cisplatin or carboplatin to a transiodoplatin (PtI{sub 2}X{sub 2}) form. The binding is only at the N{sup δ} atom of His15. A further Pt species (PtI{sub 3}X) is also seen, in both cases bound in a crevice between symmetry-related protein molecules. Cisplatin and carboplatin are platinum anticancer agents that are used to treat a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine in hen egg-white lysozyme (HEWL) showed a partial chemical conversion of carboplatin to cisplatin owing to the high sodium chloride concentration used in the crystallization conditions. Also, the co-crystallization of HEWL with carboplatin in sodium bromide conditions resulted in the partial conversion of carboplatin to the transbromoplatin form, with a portion of the cyclobutanedicarboxylate (CBDC) moiety still present. The results of the co-crystallization of HEWL with cisplatin or carboplatin in sodium iodide conditions are now reported in order to determine whether the cisplatin and carboplatin converted to the iodo form, and whether this took place in a similar way to the partial conversion of carboplatin to cisplatin in NaCl conditions or to transbromoplatin in NaBr conditions as seen previously. It is reported here that a partial chemical transformation has taken place to a transplatin form for both ligands. The NaI-grown crystals belonged to the monoclinic space group P2{sub 1} with two molecules in the asymmetric unit. The chemically transformed cisplatin and carboplatin bind to both His15 residues, i.e. in each asymmetric unit. The binding is only at the N{sup δ} atom of His15. A third platinum species is also seen in both conditions bound in a crevice between symmetry-related molecules. Here, the platinum is bound to three I atoms identified based on their anomalous difference electron densities

  8. Extracellular vesicles in cartilage homeostasis and osteoarthritis.

    Science.gov (United States)

    Miyaki, Shigeru; Lotz, Martin K

    2018-01-01

    Extracellular vesicles carry bioactive molecules that can be transferred between cells and tissues. The purpose of this review is to describe how extracellular vesicles regulate functions of cells in cartilage and other joint tissues. The potential application of extracellular vesicles in the treatment of osteoarthritis and as biomarkers will also be discussed. Extracellular vesicles are found in synovial fluid, in articular cartilage and in the supernatants of synoviocytes and chondrocytes. Extracellular vesicles in cartilage have been proposed to be involved in cross talk between cells in joint tissues and to affect extracellular matrix turnover and inflammation. Extracellular vesicles from arthritic joints can promote abnormal gene expression and changes in cartilage extracellular matrix, including abnormal mineralization. Promising results were obtained in the therapeutic application of mesenchymal stem cell-derived extracellular vesicles for cartilage repair and experimental osteoarthritis. Extracellular vesicles have emerged as vehicles for the exchange of bioactive signaling molecules within cartilage and between joint tissues to promote joint homeostasis and arthritis pathogenesis. As the molecular content of extracellular vesicles can be customized, they offer utility in therapeutic applications.

  9. Structural basis for the sequestration of the anti-σ(70) factor Rsd from σ(70) by the histidine-containing phosphocarrier protein HPr.

    Science.gov (United States)

    Park, Young Ha; Um, Si Hyeon; Song, Saemee; Seok, Yeong Jae; Ha, Nam Chul

    2015-10-01

    Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ(70) to facilitate switching of the sigma subunit on RNA polymerase from σ(70) to the stress-responsive sigma factor σ(S) in stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ(70). The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr-Rsd interaction significantly influenced the competition between HPr and σ(70) for binding to Rsd both in vitro and in vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.

  10. Divalent metal binding by histidine-rich glycoprotein differentially regulates higher order oligomerisation and proteolytic processing.

    Science.gov (United States)

    Priebatsch, Kristin M; Poon, Ivan K H; Patel, Kruti K; Kvansakul, Marc; Hulett, Mark D

    2017-01-01

    The serum protein histidine-rich glycoprotein (HRG) has been implicated in tissue injury and tumour growth. Several HRG functions are regulated by the divalent metal Zn2+ , including ligand binding and proteolytic processing that releases active HRG fragments. Although HRG can bind divalent metals other than Zn2+ , the impact of these divalent metals on the biophysical properties of HRG remains poorly understood. We now show that HRG binds Zn2+ , Ni2+ , Cu2+ and Co2+ with micromolar affinities, but differing stoichiometries, and regulate the release of specific HRG fragments during proteolysis. Furthermore, HRG binding to Zn2+ promotes HRG dimer formation in a Zn2+ -concentration- and pH-dependent manner. Our data highlight the complex divalent metal-dependent regulatory mechanisms that govern HRG function. © 2016 Federation of European Biochemical Societies.

  11. Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

    Science.gov (United States)

    Gerth, M L; Liu, Y; Jiao, W; Zhang, X-X; Baker, E N; Lott, J S; Rainey, P B; Johnston, J M

    2017-08-01

    Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Using Poly-L-Histidine Modified Glassy Carbon Electrode to Trace Hydroquinone in the Sewage Water

    Directory of Open Access Journals (Sweden)

    Bin Wang

    2014-01-01

    Full Text Available A sensitive voltammetric method for trace measurements of hydroquinone in the sewage water is described. The poly-L-histidine is prepared to modify the glassy carbon electrode in order to improve the electrochemical catalysis of interesting substances such as hydroquinone. The influence of the base solution, pH value, and scanning speed on the tracing of hydroquinone is discussed, and the experimental procedures and conditions are optimized. The laboratory results show that it is possible to construct a linear calibration curve between the peak current of hydroquinone on modified electrode and its concentration at the level of 0.00001 mol/L. The potential limitation of the method is suggested by a linear peaking shift model as well. The method was successfully applied to the determination of hydroquinone in the actual sample of industrial waste water.

  13. Quantum Chemical Mass Spectrometry: Verification and Extension of the Mobile Proton Model for Histidine

    Science.gov (United States)

    Cautereels, Julie; Blockhuys, Frank

    2017-06-01

    The quantum chemical mass spectrometry for materials science (QCMS2) method is used to verify the proposed mechanism for proton transfer - the Mobile Proton Model (MPM) - by histidine for ten XHS tripeptides, based on quantum chemical calculations at the DFT/B3LYP/6-311+G* level of theory. The fragmentations of the different intermediate structures in the MPM mechanism are studied within the QCMS2 framework, and the energetics of the proposed mechanism itself and those of the fragmentations of the intermediate structures are compared, leading to the computational confirmation of the MPM. In addition, the calculations suggest that the mechanism should be extended from considering only the formation of five-membered ring intermediates to include larger-ring intermediates. [Figure not available: see fulltext.

  14. Deep-vein thrombosis is not associated with the P/S186 polymorphism of histidine-rich glycoprotein

    NARCIS (Netherlands)

    Rattink, A.P.; Hennis, B.C.; Lievers, C.J.A.; Maat, M.P.M. de; Bertina, R.; Mennen, L.I.; Rosendaal, F.R.

    1999-01-01

    Background: In several studies, higher plasma levels of histidine-rich glycoprotein (HRG) have been observed in patients with venous thrombosis than in healthy subjects. Apart from environmental factors, such as the use of oral contraceptives, the plasma HRG levels are mainly determined genetically.

  15. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    Science.gov (United States)

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  16. Implication of citrate, malate and histidine in the accumulation and transport of nickel in Mesembryanthemum crystallinum and Brassica juncea.

    Science.gov (United States)

    Amari, Taoufik; Lutts, Stanley; Taamali, Manel; Lucchini, Giorgio; Sacchi, Gian Attilio; Abdelly, Chedly; Ghnaya, Tahar

    2016-04-01

    Citrate, malate and histidine have been involved in many processes including metal tolerance and accumulation in plants. These molecules have been frequently reported to be the potential nickel chelators, which most likely facilitate metal transport through xylem. In this context, we assess here, the relationship between organics acids and histidine content and nickel accumulation in Mesembryanthemum crystallinum and Brassica juncea grown in hydroponic media added with 25, 50 and 100 µM NiCl2. Results showed that M. crystallinum is relatively more tolerant to Ni toxicity than B. juncea. For both species, xylem transport rate of Ni increased with increasing Ni supply. A positive correlation was established between nickel and citrate concentrations in the xylem sap. In the shoot of B. juncea, citric and malic acids concentrations were significantly higher than in the shoot of M. crystallinum. Also, the shoots and roots of B. juncea accumulated much more histidine. In contrast, a higher root citrate concentration was observed in M. crystallinum. These findings suggest a specific involvement of malic and citric acid in Ni translocation and accumulation in M. crystallinum and B. juncea. The high citrate and histidine accumulation especially at 100µM NiCl2, in the roots of M. crystallinum might be among the important factors associated with the tolerance of this halophyte to toxic Ni levels. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. High-energy collision-induced dissociation of histidine ions, [His+H](+) and [His-H](-) , and histidine dimer [His2 +H]().

    Science.gov (United States)

    Khreis, Jusuf M; Reitshammer, Julia; Vizcaino, Violaine; Klawitter, Kevin; Feketeová, Linda; Denifl, Stephan

    2017-11-06

    Histidine (His) is an essential amino acid, whose side group consists of aromatic imidazole moiety that can bind a proton or metal cation and act as a donor in intermolecular interactions in many biological processes. While the dissociation of His monomer ions is well known, information on the kinetic energy released in the dissociation has been missing. Using a new home built electrospray ionization (ESI) source adapted to a double focusing mass spectrometer of BxE geometry, we investigated the fragmentation reactions of protonated and deprotonated His, [His+H](+) and [His-H](-) , and the protonated His dimer [His2 +H](+) , accelerated to 6 keV in a high-energy collision with He gas. We have evaluated the kinetic energy release (KER) for the observed dissociation channels. ESI of the His solution in positive mode led to the formation of His clusters [Hisn +H](+) , n = 1 - 6, with notably enhanced stability of the tetramer. [His+H](+) dissociates predominantly by loss of (H2 O+CO) with a KER of 278 meV, while the dominant dissociation channel of [His-H](-) involves loss of NH3 with a high KER of 769 meV. Dissociation of [His2 +H](+) is dominated by loss of the monomer but smaller losses are also observed. The KER for HCOOH loss from both [His+H](+) and [His-H](-) is similar at 278 and 249 meV, respectively, which suggest the collision-induced dissociation takes place via a similar mechanism. The loss of COOH and C2 H5 NO2 from the dimer suggests that the dimer of His binds through a shared proton between the imidazole moieties. This article is protected by copyright. All rights reserved.

  18. A Novel Protective Function for Cytokinin in the Light Stress Response Is Mediated by the ARABIDOPSIS HISTIDINE KINASE2 and ARABIDOPSIS HISTIDINE KINASE3 Receptors1[W

    Science.gov (United States)

    Cortleven, Anne; Nitschke, Silvia; Klaumünzer, Marion; AbdElgawad, Hamada; Asard, Han; Grimm, Bernhard; Riefler, Michael; Schmülling, Thomas

    2014-01-01

    Cytokinins are plant hormones that regulate diverse processes in plant development and responses to biotic and abiotic stresses. In this study, we show that Arabidopsis (Arabidopsis thaliana) plants with a reduced cytokinin status (i.e. cytokinin receptor mutants and transgenic cytokinin-deficient plants) are more susceptible to light stress compared with wild-type plants. This was reflected by a stronger photoinhibition after 24 h of high light (approximately 1,000 µmol m−2 s−1), as shown by the decline in maximum quantum efficiency of photosystem II photochemistry. Photosystem II, especially the D1 protein, is highly sensitive to the detrimental impact of light. Therefore, photoinhibition is always observed when the rate of photodamage exceeds the rate of D1 repair. We demonstrate that in plants with a reduced cytokinin status, the D1 protein level was strongly decreased upon light stress. Inhibition of the D1 repair cycle by lincomycin treatment indicated that these plants experience stronger photodamage. The efficiency of photoprotective mechanisms, such as nonenzymatic and enzymatic scavenging systems, was decreased in plants with a reduced cytokinin status, which could be a cause for the increased photodamage and subsequent D1 degradation. Additionally, slow and incomplete recovery in these plants after light stress indicated insufficient D1 repair. Mutant analysis revealed that the protective function of cytokinin during light stress depends on the ARABIDOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 receptors and the type B ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) and ARR12. We conclude that proper cytokinin signaling and regulation of specific target genes are necessary to protect leaves efficiently from light stress. PMID:24424319

  19. A novel protective function for cytokinin in the light stress response is mediated by the Arabidopsis histidine kinase2 and Arabidopsis histidine kinase3 receptors.

    Science.gov (United States)

    Cortleven, Anne; Nitschke, Silvia; Klaumünzer, Marion; Abdelgawad, Hamada; Asard, Han; Grimm, Bernhard; Riefler, Michael; Schmülling, Thomas

    2014-03-01

    Cytokinins are plant hormones that regulate diverse processes in plant development and responses to biotic and abiotic stresses. In this study, we show that Arabidopsis (Arabidopsis thaliana) plants with a reduced cytokinin status (i.e. cytokinin receptor mutants and transgenic cytokinin-deficient plants) are more susceptible to light stress compared with wild-type plants. This was reflected by a stronger photoinhibition after 24 h of high light (approximately 1,000 µmol m(-2) s(-1)), as shown by the decline in maximum quantum efficiency of photosystem II photochemistry. Photosystem II, especially the D1 protein, is highly sensitive to the detrimental impact of light. Therefore, photoinhibition is always observed when the rate of photodamage exceeds the rate of D1 repair. We demonstrate that in plants with a reduced cytokinin status, the D1 protein level was strongly decreased upon light stress. Inhibition of the D1 repair cycle by lincomycin treatment indicated that these plants experience stronger photodamage. The efficiency of photoprotective mechanisms, such as nonenzymatic and enzymatic scavenging systems, was decreased in plants with a reduced cytokinin status, which could be a cause for the increased photodamage and subsequent D1 degradation. Additionally, slow and incomplete recovery in these plants after light stress indicated insufficient D1 repair. Mutant analysis revealed that the protective function of cytokinin during light stress depends on the Arabidopsis histidine KINASE2 (AHK2) and AHK3 receptors and the type B Arabidopsis response regulator1 (ARR1) and ARR12. We conclude that proper cytokinin signaling and regulation of specific target genes are necessary to protect leaves efficiently from light stress.

  20. Novel mutations and clinical outcomes of copper-histidine therapy in Menkes disease patients.

    Science.gov (United States)

    Kim, Ja Hye; Lee, Beom Hee; Kim, Yoo-Mi; Choi, Jin-Ho; Kim, Gu-Hwan; Cheon, Chong Kun; Yoo, Han-Wook

    2015-02-01

    Menkes disease is a very rare X-linked copper metabolism disorder that results from an ATP7A gene mutation. With the advent of subcutaneous copper-histidine therapy, the early diagnosis of Menkes disease becomes of utmost importance for patients' prognosis. In the present study, the clinical characteristics of 12 Korean patients with Menkes disease (11 males and 1 female from 11 unrelated families) were described along with the mutation spectrum. Only 2 male patients were diagnosed in the neonatal period, and the other male patients were diagnosed at age 4.3 ± 1.9 months. The presenting signs included depigmented kinky hair, neurologic deficits, and hypotonia. Serum copper and ceruloplasmin levels were markedly decreased. Intracranial vessels were dilated with tortuosity and accompanied by regional cerebral infarctions, even at an early age. Of note, the female patient was diagnosed at age 18 months, during the evaluation for developmental delay, by characteristic MRA findings, biochemical profiles, and genetic evaluation. A total of 11 ATP7A mutations were identified, including five previously unreported mutations. Most mutations were truncated (except 1 missense mutation), including 3 frameshift, 2 nonsense, 3 large deletion, and 2 splice-site variants. The age at commencement of copper-histidine treatment was variable among patients age 7.3 ± 7.5 (0.5-27) months. Despite the treatment, seven patients died before age 5 years, and the remaining patients were severely retarded in neurodevelopment. The poor outcomes of our patients might be related to delayed therapy, but severe ATP7A mutations should be noted as well.

  1. Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography.

    Science.gov (United States)

    Dong, Xiao-Yan; Chen, Li-Jun; Sun, Yan

    2009-07-03

    This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and beta-cyclodextrin (beta-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein-CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing beta-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6-0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.

  2. In silico study of fragile histidine triad interaction domains with MDM2 and p53

    Directory of Open Access Journals (Sweden)

    Ameneh Eslamparast

    2014-01-01

    Full Text Available Background: Fragile histidine triad (FHIT is considered as a member of the histidine triad (HIT nucleotide-binding protein superfamily regarded as a putative tumor suppressor executing crucial role in inhibiting p53 degradation by MDM2. Accumulating evidences indicate FHIT interaction with p53 or MDM2; however, there is no certain study deciphering functional domains of FHIT involving in the interaction with MDM2 and/or p53. In this regard, such evident interaction can spring in mind determining important domains of FHIT binding to MDM2 with regard to p53. Materials and Methods: Since there were not any previous studies appraising complete three-dimensional structures of target molecules, molecular modeling was carried out to construct three-dimensional models of full FHIT, MDM2, P53 and also FHIT segments. Truncated structures of FHIT were created to reveal critical regions engaging in FHIT interaction. Results: Given the shape and shape/electrostatic total energy, FHIT structures (β1-5, (β3-7, α1, and (β5-7, α1 appeared to be better candidates than other structures in interaction with full MDM2. Furthermore, FHIT structures (β6-7, (β6-7, α1, (β4-7, α1 were considered to be better than other structures in interaction with p53. FHIT truncates that interact with MDM2 presented lower energy levels than FHIT truncates interacting with p53. Conclusion: These findings are beneficial to understand the mechanism of the FHIT-MDM2-p53 complex activation for designing inhibitory compounds.

  3. Extracellular nucleotide signaling in plants

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  4. Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693

    DEFF Research Database (Denmark)

    Martinez, Virginia; de Santos, Patricia Gómez; García-Hidalgo, Javier

    2015-01-01

    Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidin......Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine......, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main...... reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3...

  5. Involvement of disulfide bonds and histidine 172 in a unique beta-sheet to alpha-helix transition of alpha 1-acid glycoprotein at the biomembrane interface.

    Science.gov (United States)

    Nishi, Koji; Komine, Yoshio; Fukunaga, Naoko; Maruyama, Toru; Suenaga, Ayaka; Otagiri, Masaki

    2006-05-15

    Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes. (c) 2006 Wiley-Liss, Inc.

  6. Loss of the histidine kinase DhkD results in mobile mounds during development of Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Charles K Singleton

    Full Text Available Histidine kinases are receptors for sensing cellular and environmental signals, and in response to the appropriate cue they initiate phosphorelays that regulate the activity of response regulators. The Dictyostelium discoideum genome encodes 15 histidine kinases that function to regulate several processes during the multicellular developmental program, including the slug to culmination transition, osmoregulation, and spore differentiation. While there are many histidine kinases, there is only a single response regulator, RegA. Not surprisingly given the ubiquitous involvement of cAMP in numerous processes of development in Dictyostelium, RegA is a cAMP phosphodiesterase that is activated upon receiving phosphates through a phosphorelay. Hence, all of the histidine kinases characterized to date regulate developmental processes through modulating cAMP production. Here we investigate the function of the histidine kinase DhkD.The dhkD gene was disrupted, and the resulting cells when developed gave a novel phenotype. Upon aggregation, which occurred without streaming, the mounds were motile, a phenotype termed the pollywog stage. The pollywog phenotype was dependent on a functional RegA. After a period of random migration, the pollywogs attempted to form fingers but mostly generated aberrant structures with no tips. While prestalk and prespore cell differentiation occurred with normal timing, proper patterning did not occur. In contrast, wild type mounds are not motile, and the cAMP chemotactic movement of cells within the mound facilitates proper prestalk and prespore patterning, tip formation, and the vertical elongation of the mound into a finger.We postulate that DhkD functions to ensure the proper cAMP distribution within mounds that in turn results in patterning, tip formation and the transition of mounds to fingers. In the absence of DhkD, aberrant cell movements in response to an altered cAMP distribution result in mound migration, a lack of

  7. Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii.

    Science.gov (United States)

    Foureau, Emilien; Clastre, Marc; Montoya, Erika J Obando; Besseau, Sébastien; Oudin, Audrey; Glévarec, Gaëlle; Simkin, Andrew J; Crèche, Joël; Atehortùa, Lucia; Giglioli-Guivarc'h, Nathalie; Courdavault, Vincent; Papon, Nicolas

    2014-04-01

    Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    Science.gov (United States)

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  9. Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

    Directory of Open Access Journals (Sweden)

    Mimi Yang

    2015-06-01

    Full Text Available Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30 mg/L and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1. PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture

  10. Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73

    OpenAIRE

    Kim, Yong Hwan; Park, Seur Kee; Hur, Jin Young; Kim, Young Cheol

    2017-01-01

    Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin typ...

  11. Extracellular Molecules Involved in Cancer Cell Invasion

    Energy Technology Data Exchange (ETDEWEB)

    Stivarou, Theodora; Patsavoudi, Evangelia, E-mail: epatsavoudi@pasteur.gr [Department of Biochemistry, Hellenic Pasteur Institute, Athens 11521 (Greece); Technological Educational Institute of Athens, Egaleo, Athens 12210 (Greece)

    2015-01-26

    Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  12. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  13. Extracellular polymeric substances act as transient media in extracellular electron transfer of Shewanella oneidensis MR-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, Jingdong; Ulstrup, Jens

    It is well known that microorganism is surrounded by extracellular polymeric substances (EPS) which include polysaccharides, proteins, glycoproteins, nucleic acids, phospholipids, and humic acids. However, previous studies on microbial extracellular electron transfer (EET) are conducted on cells...

  14. Extracellular vesicles and blood diseases.

    Science.gov (United States)

    Nomura, Shosaku

    2017-04-01

    Extracellular vesicles (EVs) are small membrane vesicles released from many different cell types by the exocytic budding of the plasma membrane in response to cellular activation or apoptosis. EVs disseminate various bioactive effectors originating from the parent cells and transfer functional RNA and protein between cells, enabling them to alter vascular function and induce biological responses involved in vascular homeostasis. Although most EVs in human blood originate from platelets, EVs are also released from leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells. EVs were initially thought to be small particles with procoagulant activity; however, they can also evoke cellular responses in the immediate microenvironments and transport microRNAs (miRNA) into target cells. In this review, we summarize the recent literature relevant to EVs, including a growing list of clinical disorders that are associated with elevated EV levels. These studies suggest that EVs play roles in various blood diseases.

  15. Assembly of Fibronectin Extracellular Matrix

    Science.gov (United States)

    Singh, Purva; Carraher, Cara; Schwarzbauer, Jean E.

    2013-01-01

    In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5β1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation. PMID:20690820

  16. Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum.

    Science.gov (United States)

    Suess, Patrick M; Gomer, Richard H

    2016-09-16

    Polyphosphate is a polymer of phosphate residues linked by high energy phosphoanhydride bonds. Despite being highly conserved throughout nature, its function is poorly understood. Here we show that Dictyostelium cells accumulate extracellular polyphosphate, and this acts to inhibit proliferation at high cell densities. In shaking culture, extracellular polyphosphate concentrations increase as cell density increases, and if the concentration of polyphosphate observed at the stationary phase is added to cells at mid-log, proliferation is halted. Adding an exopolyphosphatase to cell cultures or stationary phase conditioned medium decreases polyphosphate levels and abrogates the anti-proliferative effect. The cells show saturable binding of polyphosphate, suggesting the presence of a cell surface polyphosphate receptor. Extracellular polyphosphate accumulation is potentiated by decreased nutrient levels, potentially as a means to anticipate starvation. Loss of the Dictyostelium polyphosphate kinase DdPpk1 causes intracellular polyphosphate levels to become undetectable and negatively affects fitness, cytokinesis, and germination. However, cells lacking DdPpk1 accumulate ∼50% normal levels of extracellular polyphosphate, suggesting an additional means of synthesis. We found that cells lacking inositol hexakisphosphate kinase, which is responsible for the synthesis of the inositol pyrophosphates IP7 and IP8, reach abnormally high cell densities and show decreased extracellular polyphosphate levels. Two different enzymes thus appear to mediate the synthesis of Dictyostelium extracellular polyphosphate, which is used as a signal in an autocrine negative feedback loop to regulate cell proliferation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Dietary histidine requirement to reduce the risk and severity of cataracts is higher than the requirement for growth in Atlantic salmon smolts, independently of the dietary lipid source.

    Science.gov (United States)

    Remø, S C; Hevrøy, E M; Olsvik, P A; Fontanillas, R; Breck, O; Waagbø, R

    2014-05-28

    The present study was carried out to investigate whether the dietary histidine requirement to reduce cataract development is higher than that for growth in Atlantic salmon smolts (Salmo salar L.) after seawater transfer and whether dietary vegetable oils contribute to cataractogenesis. Duplicate groups of salmon smolts were fed ten experimental diets with either fish oil (FO) or a vegetable oil (VO) mix replacing 70 % FO and histidine at five target levels (10, 12, 14, 16 and 18 g His/kg diet) for 13 weeks after seawater transfer. The VO diet-fed fish exhibited somewhat inferior growth and feed intakes compared with the FO diet-fed fish, irrespective of the dietary histidine concentration. Both cataract prevalence and severity were negatively correlated with the dietary histidine concentration, while lens N-acetyl-histidine (NAH) concentrations were positively correlated with it. The fatty acid profiles of muscle, heart and lens reflected that of the dietary oils to a descending degree and did not affect the observed cataract development. Muscle, heart and brain histidine concentrations reflected dietary histidine concentrations, while the corresponding tissue imidazole (anserine, carnosine and NAH) concentrations appeared to saturate differently with time. The expression level of liver histidase was not affected by the dietary histidine concentration, while the liver antioxidant response was affected in the VO diet-fed fish on a transcriptional level. The lowest severity of cataracts could be achieved by feeding 13·4 g His/kg feed, independently of the dietary lipid source. However, the present study also suggests that the dietary histidine requirement to minimise the risk of cataract development is 14·4 g His/kg feed.

  18. Illuminating the physiology of extracellular vesicles

    OpenAIRE

    Choi, Hongyoon; Lee, Dong Soo

    2016-01-01

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for trackin...

  19. Optimization of extracellular catalase production from Aspergillus ...

    African Journals Online (AJOL)

    aghomotsegin

    somes but not usually excreted from the cell. Industrially, the extracellular .... aConcentration (g/L) ; bConcentration (mg/L); CDW, cell dry weight ; E.A, extracellular catalase activity ; LL, low level; HL, high level. was used for the ..... and their relationship to other eukaryotic and prokaryotic catalases. J. Mol. Evol. 42(5): ...

  20. Detection of extracellular vesicles: size does matter

    NARCIS (Netherlands)

    van der Pol, E.

    2015-01-01

    Cells release small sacks filled with fluid, which are called "extracellular vesicles". The diameter of extracellular vesicles (EV) typically ranges from 30 nm to 1 µm. Because cells release EV into their environment, our body fluids contain numerous EV. Cells release EV to remove waste and to

  1. Urinary extracellular vesicles: biomarkers and beyond

    NARCIS (Netherlands)

    M. Salih (Mahdi)

    2017-01-01

    markdownabstractExtracellular vesicles have been isolated in various body fluids including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly the pathophysiological state of cells lining the nephron. Because

  2. Oxidative Stress Tolerance by Calcium and Histidine in Two Tomato Cultivars Under Nickel Stress

    Directory of Open Access Journals (Sweden)

    Mozafari H.

    2014-05-01

    Full Text Available We investigated calcium (Ca and L-histidine (His interaction on nickel (Ni-induced oxidative stress tolerance in two tomato (Solanum lycopersicum Mill. cultivars including Cal-J N3 and Petoearly CH. CaCl2 (0 and 300 µM and L-histidine (0 and 300 µM effects on the oxidative responses in these cultivars cultured were compared in the hydroponic media under Ni stress (NiSO4; 0,150 and 300 µM. The activities of antioxidative enzymes including catalase (CAT, guaiacol peroxidase (GPX, ascorbate peroxidase (APX, superoxide dismutase (SOD and total content of proteins, malondialdehyde (MDA, other aldehydes, H2O2, Ca2+, Ni2+, ascorbate (ASC, dehydroascorbate (DHA and electrolytes leakage (EL were determined. The obtained results indicated that the application of Ca and His generally reduced oxidative markers such as the contents of EL, H2O2, MDA and activity of CAT as well as the Ni2+content of root and shoot organs under nickel toxicity, while application of Ni treatment without Ca+His increased these oxidative parameters and accumulation of Ni2+, compared to the control. Applying Ni without Ca and His has resulted in reduction of GPX, APX and SOD activities as well as concentrations of root and shoot Ca2+and ASC in the two mentioned cultivars. Application of Ca and His lead to the elevated contents of Ca2+ and ASC, increased activities of GPX, APX and SOD as well as inhibition of Ni2+ accumulation differently in both cultivars. Ca and His also alleviated the adverse effects of Ni stress on the selected investigated parameters especially in Petoearly CH cultivar. Thus, interaction of Ca and His appeared to improve adaptive responses to Ni stress leading to decreasing Ni-induced oxidative stress in the tomato plants. Therefore, our results suggest that Ca+His alleviated nickel-induced oxidative stress by uptake and inhibition of translocation of Ni2+ plus Ni chelating mechanism improvement in the tomato cultivars.

  3. Metabolic profiling of plasma amino acids shows that histidine increases following the consumption of pork

    Directory of Open Access Journals (Sweden)

    Samman S

    2014-06-01

    Full Text Available Samir Samman,1 Ben Crossett,2 Miles Somers,1 Kirstine J Bell,1 Nicole T Lai,1,3 David R Sullivan,3 Peter Petocz4 1Discipline of Nutrition and Metabolism, 2Discipline of Proteomics and Biotechnology, School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia; 3Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Sydney, NSW, Australia; 4Department of Statistics, Macquarie University, Sydney, NSW, Australia Abstract: Amino acid (AA status is determined by factors including nutrition, metabolic rate, and interactions between the metabolism of AA, carbohydrates, and lipids. Analysis of the plasma AA profile, together with markers of glucose and lipid metabolism, will shed light on metabolic regulation. The objectives of this study were to investigate the acute responses to the consumption of meals containing either pork (PM or chicken (CM, and to identify relationships between plasma AA and markers of glycemic and lipemic control. A secondary aim was to explore AA predictors of plasma zinc concentrations. Ten healthy adults participated in a postprandial study on two separate occasions. In a randomized cross-over design, participants consumed PM or CM. The concentrations of 21 AA, glucose, insulin, triglycerides, nonesterified fatty acids, and zinc were determined over 5 hours postprandially. The meal composition did not influence glucose, insulin, triglyceride, nonesterified fatty acid, or zinc concentrations. Plasma histidine was higher following the consumption of PM (P=0.014, with consistently higher changes observed after 60 minutes (P<0.001. Greater percentage increases were noted at limited time points for valine and leucine + isoleucine in those who consumed CM compared to PM. In linear regression, some AAs emerged as predictors of the metabolic responses, irrespective of the meal that was consumed. The present study demonstrates that a single meal of PM or CM produces a differential profile of AA in the

  4. Solution Equilibria between Aluminum(III) Ion and L-histidine or L-tyrosine

    Science.gov (United States)

    Jelic, Ratomir; Dzajevic, Dragana; Cvijovic, Mirjana

    2002-01-01

    Toxic effects due to high aluminum body loads were observed in a number of conditions following ingestion of Al-containing antacids. Bio-availability of aluminum depends not only on the solubility of the ingested salt but also on the physico-chemical properties of the soluble Al complexes formed in body fluids. Amino acids may, upon interaction with Al-salts, form absorbable Al-complexes. Hence, complex formation equilibria between Al3+ and either, L- histidine or L-tyrosine were studied by glass electrode potentiometric (0.1 mol/L LiCl ionic medium, 298 K), proton NMR and uv spectrophotometric measurements. Non linear least squares treatment of the potentiometric data indicates that in the concentration ranges: 0.5≤CA1≤2.0 ; 1.0≤CHis≤10.0; 2.5≤PH≤6.5, in Al3+ + His solutions, the following complexes (with log overall stability constants given in parenthesis) are formed: Al(HHis)3+(12.21±0.08); Al(His)2+, (7.25±0.08); and Al(HHis)His2+, (20.3±0.1). In Al3+ + Tyr solutions in the concentration range 1.0≤CTyr≤3.0 mmol/L and ligand to metal concentration ratio from 2:1 to 3:1, in the pH interval from 3.0 to 6.5 the formation of the following complexes was detected: Al(HTyr)2+, (12.72±0.09); Al(Tyr)2+, (10.16±0.03) and Al(OH)2Tyr , (2.70±0.05). Proton NMR data indicate that in Al(His)2+ complex histidine acts as a monodentate ligand but its bidentate coordination is possible with carboxylate oxygen and imidazole 1-nitrogen as donors. In Al(HTyr)3+ complex tyrosine is a monodentate ligand with carboxylate oxygen as donor. The mechanism of the formation of complexes in solution is discussed as well as their possible role in aluminum toxicity. PMID:18476000

  5. Xenogenic extracellular matrices as potential biomaterials for interposition grafting in urological surgery.

    LENUS (Irish Health Repository)

    Davis, N F

    2012-01-31

    PURPOSE: The field of tissue engineering focuses on developing strategies for reconstructing injured, diseased, and congenitally absent tissues and organs. During the last decade urologists have benefited from remodeling and regenerative properties of bioscaffolds derived from xenogenic extracellular matrices. We comprehensively reviewed the current literature on structural and functional characteristics of xenogenic extracellular matrix grafting since it was first described in urological surgery. We also reviewed the clinical limitations, and assessed the potential for safe and effective urological application of extracellular matrix grafting in place of autogenous tissue. MATERIALS AND METHODS: We performed literature searches for English language publications using the PubMed(R) and MEDLINE(R) databases. Keywords included "xenogenic," "extracellular matrix" and "genitourinary tract applications." A total of 112 articles were scrutinized, of which 50 were suitable for review based on clinical relevance and importance of content. RESULTS: Since the mid 1990s xenogenic extracellular matrices have been used to successfully treat a number of pathological conditions that affect the upper and lower genitourinary tract. They are typically prepared from porcine organs such as small intestine and bladder. These organs are harvested and subjected to decellularization and sterilization techniques before surgical implantation. Bioinductive growth factors that are retained during the preparation process induce constructive tissue remodeling as the extracellular matrix is simultaneously degraded and excreted. However, recent documented concerns over durability, decreased mechanical strength and residual porcine DNA after preparation techniques have temporarily hampered the potential of extracellular matrices as a reliable replacement for genitourinary tract structures. CONCLUSIONS: Extracellular matrices are a useful alternative for successfully treating a number of urological

  6. Xenogenic extracellular matrices as potential biomaterials for interposition grafting in urological surgery.

    Science.gov (United States)

    Davis, N F; McGuire, B B; Callanan, A; Flood, H D; McGloughlin, T M

    2010-12-01

    The field of tissue engineering focuses on developing strategies for reconstructing injured, diseased, and congenitally absent tissues and organs. During the last decade urologists have benefited from remodeling and regenerative properties of bioscaffolds derived from xenogenic extracellular matrices. We comprehensively reviewed the current literature on structural and functional characteristics of xenogenic extracellular matrix grafting since it was first described in urological surgery. We also reviewed the clinical limitations, and assessed the potential for safe and effective urological application of extracellular matrix grafting in place of autogenous tissue. We performed literature searches for English language publications using the PubMed® and MEDLINE® databases. Keywords included "xenogenic," "extracellular matrix" and "genitourinary tract applications." A total of 112 articles were scrutinized, of which 50 were suitable for review based on clinical relevance and importance of content. Since the mid 1990s xenogenic extracellular matrices have been used to successfully treat a number of pathological conditions that affect the upper and lower genitourinary tract. They are typically prepared from porcine organs such as small intestine and bladder. These organs are harvested and subjected to decellularization and sterilization techniques before surgical implantation. Bioinductive growth factors that are retained during the preparation process induce constructive tissue remodeling as the extracellular matrix is simultaneously degraded and excreted. However, recent documented concerns over durability, decreased mechanical strength and residual porcine DNA after preparation techniques have temporarily hampered the potential of extracellular matrices as a reliable replacement for genitourinary tract structures. Extracellular matrices are a useful alternative for successfully treating a number of urological conditions that affect the genitourinary tract. However

  7. Extracellular vesicles: new players in cardiovascular diseases.

    Science.gov (United States)

    Gaceb, Abderahim; Martinez, Maria Carmen; Andriantsitohaina, Ramaroson

    2014-05-01

    Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Homo- and Heteroligand Nickel(II Complexes with Benzoic and para-Methoxybenzoic Acid Hydrazides and L-Histidine

    Directory of Open Access Journals (Sweden)

    N.V. Troshanin

    2017-03-01

    Full Text Available Complex formation of nickel(II with benzoic, para-methoxybenzoic acid hydrazides, and L-histidine have been studied by the methods of pH-metric titrimetry, spectrophotometry, and mathematical modelling in aqueous solutions with 1.0 mol dm–3 KNO3 as background at 298 K. Dissociation constants of ligands, as well as composition, formation constants, and spectral parameters of homo- and heteroligand complexes have been determined. It has been shown that stability of the complexes formed with para-methoxybenzoic acid hydrazide is higher than with benzoic acid hydrazide, which is consistent with the electron-donor properties of the methoxy group. Extra stabilization of the nickel(II heteroligand complexes with benzoic (para-methoxybenzoic acid hydrazide and L-histidine has been discovered and interpreted.

  9. Extracellular Matrix and Liver Disease

    Science.gov (United States)

    Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier; Varela-Rey, Marta; Pérez de Obanos, María Pilar; Leung, Tung Ming; Lopategi, Aritz; Benedicto, Aitor; Abraham-Enachescu, Ioana

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. Critical Issues: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Recent Advances: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF’ apoptosis, senescence, and reversal to quiescence. Future Directions: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new “omics” tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs. Antioxid. Redox Signal. 21, 1078–1097. PMID:24219114

  10. Extracellular Control of Limb Regeneration

    Science.gov (United States)

    Calve, S.; Simon, H.-G.

    Adult newts possess the ability to completely regenerate organs and appendages. Immediately after limb loss, the extracellular matrix (ECM) undergoes dramatic changes that may provide mechanical and biochemical cues to guide the formation of the blastema, which is comprised of uncommitted stem-like cells that proliferate to replace the lost structure. Skeletal muscle is a known reservoir for blastema cells but the mechanism by which it contributes progenitor cells is still unclear. To create physiologically relevant culture conditions for the testing of primary newt muscle cells in vitro, the spatio-temporal distribution of ECM components and the mechanical properties of newt muscle were analyzed. Tenascin-C and hyaluronic acid (HA) were found to be dramatically upregulated in the amputated limb and were co-expressed around regenerating skeletal muscle. The transverse stiffness of muscle measured in situ was used as a guide to generate silicone-based substrates of physiological stiffness. Culturing newt muscle cells under different conditions revealed that the cells are sensitive to both matrix coating and substrate stiffness: Myoblasts on HA-coated soft substrates display a rounded morphology and become more elongated as the stiffness of the substrate increases. Coating of soft substrates with matrigel or fibronectin enhanced cell spreading and eventual cell fusion.

  11. Extracellular Vesicles in Cardiovascular Theranostics.

    Science.gov (United States)

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro, Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

  12. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  13. Role of histidine-related compounds to intracellular buffering in fish skeletal muscle.

    Science.gov (United States)

    Abe, H; Dobson, G P; Hoeger, U; Parkhouse, W S

    1985-10-01

    Histidine-related compounds (HRC) were analyzed in fish skeletal muscle as a means of identifying their precise role in intracellular buffering. Fish muscle was used because it contains two functionally and spatially distinct fiber types, red and white. Two fish species, rainbow trout (Salmo gairdneri) and the Pacific blue marlin (Makaira nigricans), were studied because these species demonstrate widely different activity patterns. Marlin red and white muscle buffer capacity was two times higher than trout with white muscle, buffering being two times greater than red in both species. Buffer capacity was highest in the 6.5-7.5 pH range for all tissues, which corresponded to their high anserine levels. The titrated HRC buffering was greater than the observed HRC buffering, which suggested that not all HRC were available to absorb protons. The HRC contribution to total cellular buffering varied from a high of 62% for marlin white to a low of 7% for trout red. The other principal buffers were found to be phosphate and protein with taurine contributing within red muscle in the 7.0-8.0 pH range. HRC were found to be dominant in skeletal muscle buffering by principally accounting for the buffering capacity differences found between the species and fiber types.

  14. The Rational Design, Synthesis, and Antimicrobial Properties of Thiophene Derivatives That Inhibit Bacterial Histidine Kinases.

    Science.gov (United States)

    Boibessot, Thibaut; Zschiedrich, Christopher P; Lebeau, Alexandre; Bénimèlis, David; Dunyach-Rémy, Catherine; Lavigne, Jean-Philippe; Szurmant, Hendrik; Benfodda, Zohra; Meffre, Patrick

    2016-10-13

    The emergence of multidrug-resistant bacteria emphasizes the urgent need for novel antibacterial compounds targeting unique cellular processes. Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses, are absent in mammals, and are embedded in various pathogenic pathways. To attenuate these signaling pathways, we aimed to target the TCS signal transducer histidine kinase (HK) by focusing on their highly conserved adenosine triphosphate-binding domain. We used a structure-based drug design strategy that begins from an inhibitor-bound crystal structure and includes a significant number of structurally simplifiying "intuitive" modifications to arrive at the simple achiral, biaryl target structures. Thus, ligands were designed, leading to a series of thiophene derivatives. These compounds were synthesized and evaluated in vitro against bacterial HKs. We identified eight compounds with significant inhibitory activities against these proteins, two of which exhibited broad-spectrum antimicrobial activity. The compounds were also evaluated as adjuvants for the treatment of resistant bacteria. One compound was found to restore the sensivity of these bacteria to the respective antibiotics.

  15. Histidine-rich glycoprotein can prevent development of mouse experimental glioblastoma.

    Directory of Open Access Journals (Sweden)

    Maria Kärrlander

    Full Text Available Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG, a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B, in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma.

  16. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats.

    Directory of Open Access Journals (Sweden)

    Gloria E Hoffman

    Full Text Available A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC, would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness.

  17. Acute hyponatremia after cardioplegia by histidine-tryptophane-ketoglutarate – a retrospective study

    Directory of Open Access Journals (Sweden)

    Lindner Gregor

    2012-06-01

    Full Text Available Abstract Background Hyponatremia is the most common electrolyte disorder in hospitalized patients and is known to be associated with increased mortality. The administration of antegrade single-shot, up to two liters, histidine-tryptophane-ketoglutarate (HTK solution for adequate electromechanical cardiac arrest and myocardial preservation during minimally invasive aortic valve replacement (MIAVR is a standard procedure. We aimed to determine the impact of HTK infusion on electrolyte and acid–base balance. Methods In this retrospective analysis we reviewed data on patient characteristics, type of surgery, arterial blood gas analysis during surgery and intra-/postoperative laboratory results of patients receiving surgery for MIAVR at a large tertiary care university hospital. Results A total of 25 patients were included in the study. All patients were normonatremic at start of surgery. All patients developed hyponatremia after administration of HTK solution with a significant drop of serum sodium of 15 mmol/L (p  Conclusions Acute hyponatremia during cardioplegia with HTK solution is isotonic and should probably not be corrected without presence of hypotonicity as confirmed by measurement of serum osmolality.

  18. Identification of Plasmodium falciparum isolates lacking histidine-rich protein 2 and 3 in Eritrea.

    Science.gov (United States)

    Menegon, Michela; L'Episcopia, Mariangela; Nurahmed, Abduselam M; Talha, Albadawi A; Nour, Bakri Y M; Severini, Carlo

    2017-11-01

    The histidine-rich protein 2 of Plasmodium falciparum is the most common malaria antigen targeted by rapid diagnostic tests for the specific diagnosis of P. falciparum. Recently, pfhrp2 gene deletions have been documented in P. falciparum isolates from South America and some multiple endemic countries in Africa and Asia. Parasites with such gene deletions can produce false negative diagnostic results using HRP2-based rapid diagnostic kits. In the present work, the prevalence of P. falciparum parasites lacking pfhrp2, pfhrp3, which produces a second P. falciparum antigen that is recognized by PfHRP2 -based rapid diagnostic tests, and their flanking genes was evaluated in 135 P. falciparum isolates from Gash Barka region and in 9 isolates from Debub region, in Eritrea. In the analyzed samples, 56% (81/144) of isolates were pfhrp2/pfhrp3 positive, while 9.7% (14/144) showed deletion of exon 2 of pfhrp2 gene and 43% (62/144) of isolates lacked the pfhrp3 gene. These results suggest that the pfhrp2 and pfhrp3 deletion phenomenon is present in a considerable proportion in the study areas, thus making the HRP2/3 based rapid diagnostic tests not completely reliable for malaria diagnosis in Eritrea. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Functional reconstitution of Staphylococcus aureus truncated AgrC histidine kinase in a model membrane system.

    Directory of Open Access Journals (Sweden)

    Lina Wang

    Full Text Available The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.

  20. Corticotropin-releasing hormone receptor-1 and histidine decarboxylase expression in chronic urticaria.

    Science.gov (United States)

    Papadopoulou, Nikoletta; Kalogeromitros, Demetrios; Staurianeas, Nikolaos G; Tiblalexi, Despina; Theoharides, Theoharis C

    2005-11-01

    Certain skin disorders, such as contact dermatitis and chronic urticaria, are characterized by inflammation involving mast cells and worsen by stress. The underlying mechanism of this effect, however, is not known. The skin appears to have the equivalent of a hypothalamic-pituitary-adrenal (HPA) axis, including local expression of corticotropin-releasing hormone (CRH) and its receptors (CRH-R). We have reported that acute stress and intradermal administration of CRH stimulate skin mast cells and increase vascular permeability through CRH-R1 activation. In this study, we investigated the expression of CRH-R1, the main CRH-R subtype in human skin, and the mast cell related gene histidine decarboxylase (HDC), which regulates the production of histamine, in normal and pathological skin biopsies. Quantitative real time PCR revealed that chronic urticaria expresses high levels of CRH-R1 and HDC as compared to normal foreskin, breast skin and cultured human keratinocytes. The lichen simplex samples had high expression of CRH-R1, but low HDC. These results implicate CRH-R in chronic urticaria, which is often exacerbated by stress.

  1. DNA binding and cleavage studies of copper(II) complexes with 2'-deoxyadenosine modified histidine moiety.

    Science.gov (United States)

    Borowska, Justyna; Sierant, Malgorzata; Sochacka, Elzbieta; Sanna, Daniele; Lodyga-Chruscinska, Elzbieta

    2015-09-01

    This work is focused on the study of DNA binding and cleavage properties of 2'-deoxyadenosines modified with ester/amide of histidine (his(6)dA ester, his(6)dA amide) and their copper(II) complexes. To determine the coordination mode of the complex species potentiometric and spectroscopic (UV-visible, CD, EPR) studies have been performed. The analysis of electronic absorption and fluorescence spectra has been used to find the nature of the interactions between the compounds and calf thymus DNA (CT-DNA). There is significant influence of the -NH2 and -OCH3 groups on binding of the ligands or the complexes to DNA. Only amide derivative and its complex reveal intercalative ability. In the case of his(6)dA ester and Cu(II)-his(6)dA ester the main interactions can be groove binding. DNA cleavage activities of the compounds have been examined by gel electrophoresis. The copper complexes have promoted the cleavage of plasmid DNA, but none of the ligands exhibited any chemical nuclease activity. The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.

  2. Large Variation in Detection of Histidine-Rich Protein 2 in Plasmodium falciparum Isolates from Colombia

    Science.gov (United States)

    Pava, Zuleima; Echeverry, Diego F.; Díaz, Gustavo; Murillo, Claribel

    2010-01-01

    Most rapid diagnostic tests (RDTs) available use histidine-rich protein 2 (HRP2) as a target. However, it has been reported that sequence variations of this protein affects its sensitivity. Currently, there is insufficient evidence for HRP2 variability in Plasmodium falciparum isolates from Colombia and its relationship with RDT performance. To determine possible geographic differences and their effects on the performance of RDTs, 22 blood samples from patients with P. falciparum malaria from Tumaco and Buenaventura, Colombia were assessed by measurement of HRP2 concentration by an HRP2 enzyme-linked immunosorbent assay, RDTs, and thick blood smear. Statistical analysis showed an association between RDT performance and HRP2 concentrations. No significant difference was found between locations. A large variation of antigen concentration in samples was found at same parasitemia. In contrast to previously reports, there was no correlation between initial parasitemia and HRP2 concentration. Our results indicate that antigen quantity should be studied more carefully because the sensitivity of the RDT is affected more by antigen concentration than by parasitemia. PMID:20889875

  3. Effect of Abiotic Stresses on Histidine kinases Gene Expression in Zea mays L. cv. SC. 704

    Directory of Open Access Journals (Sweden)

    Javadmanesh, Susan

    2013-02-01

    Full Text Available UV-B radiation and osmotic stress (like drought and salinity have a significant effect on physiology, morphology, biochemistry and molecular biology. To cope with such stimuli, plants must be able to effectively sense, respond to and adapt to changes in their biological activities. Hence, signal transduction pathways play important role in response to environmental stimuli. In this study, the expression of three Histidine Kinases including ZmHK1, ZmHK2 and ZmHK3a was studied in maize plants exposed to 8 days drought, salinity and UV-B stresses applying transcript approach. The semi-quantitative RT-PCR analyses of ZmHKs showed up-regulation of ZmHK1 and ZmHK3 agenes after 8 days exposure to applied stresses except salinity in leaves, although, their regulation was more prominent during drought stress. Astonishingly, exposure to these stresses showed down-regulation of all genes in maize roots. However, the ZmHK1 behavior was quite different from two other homologues and showed up-regulation in combined stresses. We suggest that ZmHK1 and ZmHK3a, as cytokinin transmembrane receptors, sense osmolarity changes in cells caused by dehydration. Our data supports the involvement of ZmHK homologues under these stresses in maize and provides a gene expression dynamics during the stress which will be valuable for further studies of the molecular mechanisms of stress tolerance in maize.

  4. Detection of histidine decarboxylase in rat aorta and cultured rat aortic smooth muscle cells.

    Science.gov (United States)

    Tippens, A S; Davis, S V; Hayes, J R; Bryda, E C; Green, T L; Gruetter, C A

    2004-08-01

    Having previously demonstrated release of histamine from mast-cell-deficient rat aorta, the objective of this study was to determine and localize histamine synthesis capability in the aorta by detecting histidine decarboxylase (HDC), the enzyme that catalyzes histamine formation. Experiments were conducted with nested reverse transcription-polymerase chain reaction (nRT-PCR) to detect HDC mRNA and with immunofluorescence and western blot analysis to detect HDC protein in rat aorta, cultured rat aortic smooth muscle (RASMC) and endothelial cells (RAEC). Gel electrophoresis of nRT-PCR products indicated HDC mRNA in liver, aorta and RASMC but not in RAEC or kidney. Sequence analysis confirmed that the band observed in RASMC was the target HDC amplicon. Immunofluorescence indicated the presence of HDC protein in RASMC and not in RAEC. Western Blot analysis revealed HDC protein (55 kDa) in liver, aorta, RASMC but not in RAEC or kidney. The results of this study are the first to demonstrate the presence of HDC mRNA and protein in rat aorta and more specifically in RASMC, indicative of their capability to synthesize histamine. Copyright 2004 Birkhäuser Verlag, Basel

  5. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

    Science.gov (United States)

    Tippens, A S; Gruetter, C A

    2004-06-01

    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  6. Effects of grain, fructose, and histidine on ruminal pH and fermentation products during an induced subacute acidosis protocol.

    Science.gov (United States)

    Golder, H M; Celi, P; Rabiee, A R; Heuer, C; Bramley, E; Miller, D W; King, R; Lean, I J

    2012-04-01

    The effects of grain, fructose, and histidine on ruminal pH and fermentation products were studied in dairy cattle during an induced subacute acidosis protocol. Thirty Holstein heifers were randomly allocated to 5 treatment groups: (1) control (no grain); (2) grain [fed at a crushed triticale dry matter intake (DMI) of 1.2% of body weight (BW)]; (3) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI); (4) grain (1.2% of BW DMI)+histidine (6 g/head); and (5) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI)+histidine (6 g/head) in a partial factorial arrangement. Heifers were fed 1 kg of grain daily with ad libitum access to ryegrass silage and alfalfa hay for 10 d. Feed was withheld for 14 h before challenge day, on which heifers were fed 200 g of alfalfa hay and then the treatment diets immediately thereafter. Rumen samples were collected 5 min after diet ingestion, 60 min later, and at 3 subsequent 50-min intervals. Grain decreased ruminal pH and increased ammonia, total volatile fatty acid (VFA), acetate, butyrate, propionate, and valerate concentrations compared with controls. The addition of grain had no effect on ruminal D- and L-lactate concentrations. Fructose markedly decreased ruminal pH and markedly increased D- and L-lactate concentrations. Fructose increased total VFA and butyrate and decreased valerate concentrations. Although histidine did not have a marked effect on ruminal fermentation, increased concentrations of histamine were observed following feeding. This study demonstrates that the substitution of some grain for fructose can lower ruminal pH and increase VFA and lactate concentrations, warranting further investigation into the role of sugars on the risk of acidosis in dairy cattle. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes

    OpenAIRE

    Larsen, R W; Nunez, D J; Morgan, W. T.; Muhoberac, B B; Ondrias, M.R.

    1992-01-01

    Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Ra...

  8. Synthesis of novel chitosan resin possessing histidine moiety and its application to the determination of trace silver by ICP-AES coupled with triplet automated-pretreatment system.

    Science.gov (United States)

    Hosoba, Minako; Oshita, Koji; Katarina, Rosi K; Takayanagi, Toshio; Oshima, Mitsuko; Motomizu, Shoji

    2009-04-20

    A novel chitosan resin, cross-linked chitosan functionalized with histidine moiety (histidine-type chitosan resin), was synthesized for the collection and concentration of trace silver in aquatic samples. A triplet automated-pretreatment system (Triplet Auto-Pret System) installed mini-columns packed with the synthesized histidine-type chitosan resin was coupled with an inductively coupled plasma-atomic emission spectrometry (ICP-AES) for a rapid and sensitive analysis. Adsorption behavior of 50 elements on the histidine-type chitosan resin was examined. A trace amount of Ag(I) was shown a good adsorption in wide pH regions (pH 5-9), and Ag(I) adsorbed was readily recovered with 1 M nitric acid solution. The limit of detection (3sigma) for silver was 0.03 microg L(-1). The system was successfully applied to river water and dipped water in silver coated container.

  9. On-line affinity selection of histidine-containing peptides using a polymeric monolithic support for capillary electrophoresis.

    Science.gov (United States)

    Vizioli, Nora M; Rusell, Maria Lucía; Carbajal, Maria Laura; Carducci, Clyde N; Grasselli, Mariano

    2005-08-01

    An on-line affinity selection method using a polymeric monolithic support is proposed for the retention of histidine-containing peptides and their subsequent separation by capillary zone electrophoresis (CZE). Monolithic capillary columns were prepared in fused-silica capillaries of 150 mum inner diameter (ID) by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iminodiacetic acid (IDA) and copper ion. Monolithic microextractors were coupled on-line near the inlet of the separation capillary (fused-silica capillary, 75 mum ID x 28 cm from the microextractor to the detector). Model peptide mixtures of histidine-containing and histidine-noncontaining peptides were assessed. Peptides were released from the sorbent by a 5 mM imidazole solution and then separated by CZE with ultraviolet detection. Relative standard deviation values for migration times and corrected peak areas were found to be lower than 5.8 and 10.5%, respectively. IDA-Cu(II) ion modified monolithic microextractors showed a chromatographic behavior and could be reused at least 25 times. The use of monolithic supports proved to be an advantageous alternative to packed particles for the preparation of microextractors.

  10. Aminooxy analog of histamine is an efficient inhibitor of mammalian L-histidine decarboxylase: combined in silico and experimental evidence.

    Science.gov (United States)

    Castro-Oropeza, R; Pino-Ángeles, A; Khomutov, M A; Urdiales, J L; Moya-García, A A; Vepsäläinen, J; Persson, L; Sarabia, F; Khomutov, A; Sánchez-Jiménez, F

    2014-03-01

    Histamine plays highlighted roles in the development of many common, emergent and rare diseases. In mammals, histamine is formed by decarboxylation of L-histidine, which is catalyzed by pyridoxal-5'-phosphate (PLP) dependent histidine decarboxylase (HDC, EC 4.1.1.22). The limited availability and stability of the protein have delayed the characterization of its structure-function relationships. Our previous knowledge on mammalian HDC, derived from both in silico and experimental approaches, indicates that an effective competitive inhibitor should be capable to form an "external aldimine-like structure" and have an imidazole group, or its proper mimetic, which provides additional affinity of PLP-inhibitor adduct to the HDC active center. This is confirmed using HEK-293 cells transfected to express human HDC and the aminooxy analog of histidine, 4(5)-aminooxymethylimidazole (O-IMHA, IC₅₀ ≈ 2 × 10(-7) M) capable to form a PLP-inhibitor complex (oxime) in the enzyme active center. Taking advantage of the availability of the human HDC X-ray structure, we have also determined the potential interactions that could stabilize this oxime in the active site of mammalian HDC.

  11. Histidine-iridium(III) coordination-based peptide luminogenic cyclization and cyclo-RGD peptides for cancer-cell targeting.

    Science.gov (United States)

    Ma, Xiaochuan; Jia, Junli; Cao, Rui; Wang, Xiaobo; Fei, Hao

    2014-12-24

    In the field of peptide drug discovery, structural constraining and fluorescent labeling are two sought-after techniques important for both basic research and pharmaceutical development. In this work, we describe an easy-to-use approach for simultaneous peptide cyclization and luminescent labeling based on iridium(III)-histidine coordination (Ir-HH cyclization). Using a series of model peptides with histidine flanking each terminus, the binding activity and reaction kinetics of Ir-HH cyclization of different ring sizes were characterized. In the series, Ir-HAnH (n = 2, 3) with moderate ring sizes provides appropriate flexibility and proper distance between histidines for cyclic formation, which leads to the best binding affinity and structural stability in physiological conditions, as compared to other Ir-HH-cyclized peptides with smaller (n = 0, 1) or larger (n = 4, 5) ring sizes. Ir-HRGDH, an Ir-HH-cyclized peptide containing integrin targeting motif Arg-Gly-Asp (RGD), showed better targeting affinity than its linear form and enhanced membrane permeability in comparison with fluorescein-labeled cyclic RGDyK peptide. Cell death inducing peptide KLA-linked Ir-HRGDH (Ir-HRGDH-KLA) showed dramatically enhanced cytotoxicity and high selectivity for cancer cells versus noncancer cells. These data demonstrate that the method conveniently combines structural constraining of peptides with luminescent imaging capabilities, which facilitates functional and intracellular characterization of potential peptide-based drug leads, thus introducing a new tool to meet emerging needs in medicinal research.

  12. Alkali metals in addition to acidic pH activate the EvgS histidine kinase sensor in Escherichia coli.

    Science.gov (United States)

    Eguchi, Yoko; Utsumi, Ryutaro

    2014-09-01

    Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Extracellular DNA metabolism in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Scott eChimileski

    2014-02-01

    Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

  14. Extracellular vesicles as emerging intercellular communicasomes.

    Science.gov (United States)

    Yoon, Yae Jin; Kim, Oh Youn; Gho, Yong Song

    2014-10-01

    All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions.

  15. Extracellular vesicles in coronary artery disease.

    Science.gov (United States)

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-05-01

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

  16. Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.

    Science.gov (United States)

    Isaev, Dmytro; Isaeva, Elena; Shatskih, Tatiana; Zhao, Qian; Smits, Nicole C; Shworak, Nicholas W; Khazipov, Rustem; Holmes, Gregory L

    2007-10-24

    The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

  17. Extracellular hydrogen peroxide produced under irradiation as the most important factor in the lethality of gamma-irradiated Paramecium tetraurelia

    Energy Technology Data Exchange (ETDEWEB)

    Croute, F.; Soleilhavoup, J.P.; Vidal, S.; Dupouy, D.; Planel, H. (Laboratoire de Biologie Medicale, 31 - Toulouse (France))

    1982-02-01

    It has been shown that the surviving fraction of ..gamma..-irradiated paramecia is correlated with the residual H/sub 2/O/sub 2/ concentration in the extracellular medium which is strongly dependent on the bacterial concentration, that is, on the enzyme content in the culture medium.

  18. Copper transporters and chaperones: Their function on ...

    Indian Academy of Sciences (India)

    2016-08-02

    Aug 2, 2016 ... membrane and intracellular vesicles (Zheng et al. ... The extracellular N- terminus has both N- and O-linked glycosylation sites at residues Asn 15 and Thr 27 respectively. There are two histidine and two methionine residues that act as copper ... followed by Cu exposure nor an increase in extracellular Cu.

  19. Immunocytochemical staining of endogenous nuclear proteins with the HIS-1 anti-poly-histidine monoclonal antibody: a potential source of error in His-tagged protein detection.

    Science.gov (United States)

    Chilumuri, Amrutha; Markiv, Anatoliy; Milton, Nathaniel G N

    2014-07-01

    Histidine-tagged proteins are widely used in biochemical studies and frequently detected with antibodies specific for the histidine tag. Immunocytochemistry is widely used in studies with overexpressed proteins to determine cellular localization and in the case of histidine-tagged proteins can be carried out with anti-polyhistidine antibodies. Recent studies have suggested that polyhistidine sequences are present within a small number of human proteins and may direct expression to the nucleus and nuclear speckles compartments of the cell. In this study immunocytochemical staining of human SH-SY5Y neuroblastoma cell lines with the HIS-1 anti-polyhistidine monoclonal antibody were determined. Results showed that the HIS-1 anti-polyhistidine monoclonal antibody stained endogenous nuclear proteins in SH-SY5Y cells. The stained proteins were contained within the nuclear membrane, but were not directly linked to DNA. In a histidine-tagged catalase overexpressing cell line the HIS-1 anti-polyhistidine monoclonal antibody showed nuclear staining, whilst staining with the CAT-505 anti-catalase monoclonal antibody showed primarily cytoplasmic staining. These results suggest that anti-polyhistidine antibody staining shows significant cross-reactivity with endogenous nuclear proteins in SH-SY5Y neuroblastoma cells and may not be suitable for localization studies of histidine-tagged proteins. Immunocytochemical studies with anti-polyhistidine antibodies and localization of histidine-tagged proteins must be confirmed with protein specific antibodies or other methodology. Copyright © 2014 Elsevier GmbH. All rights reserved.

  20. Residue N84 of yeast cystathionine beta-synthase is a determinant of reaction specificity.

    Science.gov (United States)

    Lodha, Pratik H; Hopwood, Emily M S; Manders, Adrienne L; Aitken, Susan M

    2010-07-01

    Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The kcat/KmL-Ser of the N84A, N84D and N84H mutants for the beta-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal beta-elimination activity was detected for N84D (kcat/KmL-Ser=0.23+/-0.02 M(-1) s(-1)) and N84H (kcat/KmL-Ser=0.34+/-0.06 M(-1) s(-1)), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with L-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  1. Illuminating the physiology of extracellular vesicles.

    Science.gov (United States)

    Choi, Hongyoon; Lee, Dong Soo

    2016-04-16

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for tracking and imaging extracellular vesicles in vivo and critically discuss their systemic distribution, targeting, and kinetics based on up-to-date evidence in the literature.

  2. Extracellular Vesicles in Metabolic Syndrome.

    Science.gov (United States)

    Martínez, M Carmen; Andriantsitohaina, Ramaroson

    2017-05-12

    Metabolic syndrome defines a cluster of interrelated risk factors for cardiovascular disease and diabetes mellitus. These factors include metabolic abnormalities, such as hyperglycemia, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, high blood pressure, and obesity, mainly central adiposity. In this context, extracellular vesicles (EVs) may represent novel effectors that might help to elucidate disease-specific pathways in metabolic disease. Indeed, EVs (a terminology that encompasses microparticles, exosomes, and apoptotic bodies) are emerging as a novel mean of cell-to-cell communication in physiology and pathology because they represent a new way to convey fundamental information between cells. These microstructures contain proteins, lipids, and genetic information able to modify the phenotype and function of the target cells. EVs carry specific markers of the cell of origin that make possible monitoring their fluctuations in the circulation as potential biomarkers inasmuch their circulating levels are increased in metabolic syndrome patients. Because of the mixed components of EVs, the content or the number of EVs derived from distinct cells of origin, the mode of cell stimulation, and the ensuing mechanisms for their production, it is difficult to attribute specific functions as drivers or biomarkers of diseases. This review reports recent data of EVs from different origins, including endothelial, smooth muscle cells, macrophages, hepatocytes, adipocytes, skeletal muscle, and finally, those from microbiota as bioeffectors of message, leading to metabolic syndrome. Depicting the complexity of the mechanisms involved in their functions reinforce the hypothesis that EVs are valid biomarkers, and they represent targets that can be harnessed for innovative therapeutic approaches. © 2017 American Heart Association, Inc.

  3. Comparison of the binding behavior of several histidine-containing proteins with immobilized copper(II) complexes of 1,4,7-triazacyclononane and 1,4-bis(1,4,7-triazacyclononan-1-yl)butane.

    Science.gov (United States)

    Graham, Bim; Spiccia, Leone; Hearn, Milton T W

    2011-04-15

    The protein binding characteristics of the immobilized binucleating chelate system, 1,4-bis(1,4,7-triazacyclononan-1-yl)butane (tacn(2)butane), complexed with Cu(2+) ions have been investigated with hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome C, as well as three histidine-rich proteins, serum albumin, transferrin, and α(2)-macroglobulin, present in partially fractionated human serum. The effects of pH, ionic strength and elution buffers on protein binding have been examined and compared with those of the analogous immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). The Cu(2+)-tacn(2)butane system was generally found to exhibit higher protein binding affinities than the Cu(2+)-tacn system, suggesting that the presence of immobilized binuclear copper(II) species leads to enhanced coordinative interaction with surface-exposed amino acid residues of the studied proteins. However, under some buffer conditions the dependencies of protein binding and elution on pH and ionic strength with these immobilized metal ion affinity chromatographic (IMAC) systems were consistent with electrostatic, hydrophobic and π-bonding interactions playing a significant secondary role in addition to the dominant coordinative interactions. As such, the results indicated that the selectivities were not solely dependent on the histidine content of the protein. In accord with this conclusion, differences in the selectivities of the Cu(2+)-tacn and Cu(2+)-tacn(2)butane adsorbents for serum albumin, transferrin, and α(2)-macroglobulin were observed depending on the choice of elution buffer. This attribute suggests that additional selectivity features can be realised for the separation of specific proteins with this new class of adsorbent. Copyright © 2011. Published by Elsevier B.V.

  4. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    Science.gov (United States)

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-09

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Modeling of residue stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Velasco-Arjona, A.; Castro, M.D.L. de [Univ. of Cordoba (Spain). Dept. of Analytical Chemistry; Izquierdo, A. [Gemasur, Cordoba (Spain). Dept. of R and D

    1999-04-01

    Because of the variety in anthropogenic toxic residues produced by human activity, the establishment of a general procedure for the destruction of these residues or transformation into less toxic materials is difficult. A study was made on the decrease in toxicity of various contaminated residues when treated with three different stabilizers. General criteria of the requirements for stabilization were also established, which depended on both the toxicity and nature of the toxic substances. The modeling of the stabilization process will allow nonspecialized personnel to carry out the operations and will also standardize the modus operandi of such enterprises.

  6. Extracellular polysaccharide production by Thraustochytrid protists

    Digital Repository Service at National Institute of Oceanography (India)

    Jain, R.; Raghukumar, S.; Tharanathan, R.; Bhosle, N.B.

    Four strains of marine stramenopilan protists, the thraustochytrids, were studied for their ability to produce extracellular polysaccharides (EPSs). Observations by light and scanning electron microscopy revealed the production of a matrix of EPS...

  7. Alternative methods for characterization of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Fatemeh eMomen-Heravi

    2012-09-01

    Full Text Available Extracellular vesicles are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize Extracellular vesicles. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some Extracellular vesicles -specific evidence. Characterization of Extracellular vesicles has also recently seen many advances with the use of Nanoparticle Tracking Analysis (NTA, flow cytometry, cryo-EM instruments and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.

  8. Signal Sensing and Transduction by Histidine Kinases as Unveiled through Studies on a Temperature Sensor.

    Science.gov (United States)

    Abriata, Luciano A; Albanesi, Daniela; Dal Peraro, Matteo; de Mendoza, Diego

    2017-06-20

    Histidine kinases (HK) are the sensory proteins of two-component systems, responsible for a large fraction of bacterial responses to stimuli and environmental changes. Prototypical HKs are membrane-bound proteins that phosphorylate cognate response regulator proteins in the cytoplasm upon signal detection in the membrane or periplasm. HKs stand as potential drug targets but also constitute fascinating systems for studying proteins at work, specifically regarding the chemistry and mechanics of signal detection, transduction through the membrane, and regulation of catalytic outputs. In this Account, we focus on Bacillus subtilis DesK, a membrane-bound HK part of a two-component system that maintains appropriate membrane fluidity at low growth temperatures. Unlike most HKs, DesK has no extracytoplasmic signal-sensing domains; instead, sensing is carried out by 10 transmembrane helices (coming from two protomers) arranged in an unknown structure. The fifth transmembrane helix from each protomer connects, without any of the intermediate domains found in other HKs, into the dimerization and histidine phosphotransfer (DHp) domain located in the cytoplasm, which is followed by the ATP-binding domains (ABD). Throughout the years, genetic, biochemical, structural, and computational studies on wild-type, mutant, and truncated versions of DesK allowed us to dissect several aspects of DesK's functioning, pushing forward a more general understanding of its own structure/function relationships as well as those of other HKs. We have shown that the sensing mechanism is rooted in temperature-dependent membrane properties, most likely a combination of thickness, fluidity, and water permeability, and we have proposed possible mechanisms by which DesK senses these properties and transduces the signals. X-ray structures and computational models have revealed structural features of TM and cytoplasmic regions in DesK's kinase- and phosphatase-competent states. Biochemical and genetic

  9. Extracellular Vesicles: Evolving Contributors in Autoimmunity

    OpenAIRE

    Katsiougiannis, Stergios

    2015-01-01

    Extracellular vesicles, including microvesicles, exosomes and apoptotic bodies are recognized as carriers of pathogen-associated molecules with direct involvement in immune signaling and inflammation. Those observations have enforced the way these membranous vesicles are being considered as promising immunotherapeutic targets. In this review, we discuss the emerging roles of extracellular vesicles in autoimmunity and highlights their potential use as disease biomarkers as well as targets for ...

  10. Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

    Science.gov (United States)

    Gourley, Christopher R.

    The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

  11. Histidine-Rich Glycoprotein Uptake and Turnover Is Mediated by Mononuclear Phagocytes

    Science.gov (United States)

    Tugues, Sònia; Orlova, Anna; Bhoi, Sujata; Padhan, Narendra; Åkerud, Peter; Honjo, Satoshi; Selvaraju, Ram Kumar; Mazzone, Massimiliano; Tolmachev, Vladimir; Claesson-Welsh, Lena

    2014-01-01

    Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfate-independent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread. PMID:25243896

  12. Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce.

    Science.gov (United States)

    Satomi, Masataka; Furushita, Manabu; Oikawa, Hiroshi; Yano, Yutaka

    2011-07-15

    Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006(T) were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated >99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.

    Science.gov (United States)

    Meunier, Brigitte; Maréchal, Amandine; Rich, Peter R

    2012-06-01

    Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.

  14. Histidine Regulates Seed Oil Deposition through Abscisic Acid Biosynthesis and β-Oxidation1

    Science.gov (United States)

    2016-01-01

    The storage compounds are deposited into plant seeds during maturation. As the model oilseed species, Arabidopsis (Arabidopsis thaliana) has long been studied for seed oil deposition. However, the regulation of this process remains unclear. Through genetic screen with a seed oil body-specific reporter, we isolated low oil1 (loo1) mutant. LOO1 was mapped to HISTIDINE BIOSYNTHESIS NUMBER 1A (HISN1A). HISN1A catalyzes the first step of His biosynthesis. Oil significantly decreased, and conversely proteins markedly increased in hisn1a mutants, indicating that HISN1A regulates both oil accumulation and the oil-protein balance. HISN1A was predominantly expressed in embryos and root tips. Accordingly, the hisn1a mutants exhibited developmental phenotype especially of seeds and roots. Transcriptional profiling displayed that β-oxidation was the major metabolic pathway downstream of HISN1A. β-Oxidation was induced in hisn1a mutants, whereas it was reduced in 35S:HISN1A-transgenic plants. In plants, seed storage oil is broken-down by β-oxidation, which is controlled by abscisic acid (ABA). We found that His activated genes of ABA biosynthesis and correspondingly advanced ABA accumulation. Exogenous ABA rescued the defects of hisn1a mutants, whereas mutation of ABA DEFICIENT2, a key enzyme in ABA biosynthesis, blocked the effect of His on β-oxidation, indicating that ABA mediates His regulation in β-oxidation. Intriguingly, structural analysis showed that a potential His-binding domain was present in the general amino acid sensors GENERAL CONTROL NON-DEREPRESSIBLE2 and PII, suggesting that His may serve as a signal molecule. Taken together, our study reveals that His promotes plant seed oil deposition through ABA biosynthesis and β-oxidation. PMID:27493214

  15. Histidine Regulates Seed Oil Deposition through Abscisic Acid Biosynthesis and β-Oxidation.

    Science.gov (United States)

    Ma, Huimin; Wang, Shui

    2016-10-01

    The storage compounds are deposited into plant seeds during maturation. As the model oilseed species, Arabidopsis (Arabidopsis thaliana) has long been studied for seed oil deposition. However, the regulation of this process remains unclear. Through genetic screen with a seed oil body-specific reporter, we isolated low oil1 (loo1) mutant. LOO1 was mapped to HISTIDINE BIOSYNTHESIS NUMBER 1A (HISN1A). HISN1A catalyzes the first step of His biosynthesis. Oil significantly decreased, and conversely proteins markedly increased in hisn1a mutants, indicating that HISN1A regulates both oil accumulation and the oil-protein balance. HISN1A was predominantly expressed in embryos and root tips. Accordingly, the hisn1a mutants exhibited developmental phenotype especially of seeds and roots. Transcriptional profiling displayed that β-oxidation was the major metabolic pathway downstream of HISN1A β-Oxidation was induced in hisn1a mutants, whereas it was reduced in 35S:HISN1A-transgenic plants. In plants, seed storage oil is broken-down by β-oxidation, which is controlled by abscisic acid (ABA). We found that His activated genes of ABA biosynthesis and correspondingly advanced ABA accumulation. Exogenous ABA rescued the defects of hisn1a mutants, whereas mutation of ABA DEFICIENT2, a key enzyme in ABA biosynthesis, blocked the effect of His on β-oxidation, indicating that ABA mediates His regulation in β-oxidation. Intriguingly, structural analysis showed that a potential His-binding domain was present in the general amino acid sensors GENERAL CONTROL NON-DEREPRESSIBLE2 and PII, suggesting that His may serve as a signal molecule. Taken together, our study reveals that His promotes plant seed oil deposition through ABA biosynthesis and β-oxidation. © 2016 American Society of Plant Biologists. All Rights Reserved.

  16. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis

    Science.gov (United States)

    de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Hölscher, Theresa; Kuipers, Oscar P.

    2015-01-01

    ABSTRACT Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. PMID:26152584

  17. Impact of chromium histidinate on high fat diet induced obesity in rats

    Directory of Open Access Journals (Sweden)

    Tuzcu Zeynep

    2011-05-01

    Full Text Available Abstract Background Chromium (Cr is an essential trace element that has garnered interest for use as a weight loss aid, but its molecular mechanism in obesity is not clear. In this study, an attempt has been made to investigate the effects of chromium histidinate (CrHis on glucose transporter-2 (GLUT-2, nuclear factor erythroid 2-related factor 2 (Nrf2, heme oxygenase-1 (HO-1, nuclear factor-kappa B (NF-κB p65 and the oxidative stress marker 4-hydroxynonenal adducts (HNE expressions in liver of rats fed high fat diet (HFD. Methods Male Wistar rats (n = 40, 8 wk-old were divided into four groups. Group I was fed a standard diet (12% of calories as fat; Group II was fed a standard diet and supplemented with 110 μg CrHis/kg BW/d; Group III was fed a HFD (40% of calories as fat; Group IV was fed HFD and supplemented with 110 μg CrHis/kg BW/d. Results Rats fed HFD possessed greater serum insulin (40 vs.33 pmol/L and glucose (158 vs. 143 mg/dL concentration and less liver Cr (44 vs.82 μg/g concentration than rats fed the control diet. However, rats supplemented with CrHis had greater liver Cr and serum insulin and lower glucose concentration in rats fed HFD (P P P Conclusion These findings demonstrate that supplementation of CrHis is protective against obesity, at least in part, through Nrf2-mediated induction of HO-1 in rats fed high fat diet.

  18. TENORM: Wastewater Treatment Residuals

    Science.gov (United States)

    Water and wastes which have been discharged into municipal sewers are treated at wastewater treatment plants. These may contain trace amounts of both man-made and naturally occurring radionuclides which can accumulate in the treatment plant and residuals.

  19. Production and characterization of an extracellular lipase from Candida guilliermondii

    Directory of Open Access Journals (Sweden)

    Anne Caroline Defranceschi Oliveira

    2014-12-01

    Full Text Available Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L. were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL-1 in the presence of 5 mmol L-1 NaCl at 30 ºC and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL-1. The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone and different acid:alcohol molar ratios. Higher ester conversion rate (81% was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium.

  20. Production and characterization of an extracellular lipase from Candida guilliermondii.

    Science.gov (United States)

    Oliveira, Anne Caroline Defranceschi; Fernandes, Maria Luiza; Mariano, André Bellin

    2014-01-01

    Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL(-1)) in the presence of 5 mmol L(-1) NaCl at 30 °C and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL(-1). The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone) and different acid:alcohol molar ratios. Higher ester conversion rate (81%) was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium.

  1. Purification, Chemical Characterization, and Bioactivity of an Extracellular Polysaccharide Produced by the Marine Sponge Endogenous Fungus Alternaria sp. SP-32.

    Science.gov (United States)

    Chen, Yin; Mao, Wen-Jun; Yan, Meng-Xia; Liu, Xue; Wang, Shu-Yao; Xia, Zheng; Xiao, Bo; Cao, Su-Jian; Yang, Bao-Qin; Li, Jie

    2016-06-01

    Marine sponges are ancient and simple multicellular filter-feeding invertebrates attached to solid substrates in benthic habitats and host a variety of fungi both inside and on their surface because of its unique ingestion and digest system. Investigation on marine sponge-associated fungi mainly focused on the small molecular metabolites, yet little attention had been paid to the extracellular polysaccharides. In this study, a homogeneous extracellular polysaccharide AS2-1 was obtained from the fermented broth of the marine sponge endogenous fungus Alternaria sp. SP-32 using ethanol precipitation, anion-exchange, and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that AS2-1 was composed of mannose, glucose, and galactose with a molar ratio of 1.00:0.67:0.35, and its molecular weight was 27.4 kDa. AS2-1 consists of a mannan core and a galactoglucan chain. The mannan core is composed of (1→6)-α-Manp substituted at C-2 by (1→2)-α-Manp with different degrees of polymerization. The galactoglucan chain consists of (1→6)-α-Glcp residues with (1→6)-β-Galf residues attached to the last glucopyranose residue at C-6. (1→6)-β-Galf residues have additional branches at C-2 consisting of disaccharide units of (1→2)-β-Galf and (1→2)-α-Glcp residues. The glucopyranose residue of the galactoglucan chain is linked to the mannan core. AS2-1 possessed a high antioxidant activity as evaluated by scavenging of 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals in vitro. AS2-1 was also evaluated for cytotoxic activity on Hela, HL-60, and K562 cell lines by the MTT and SRB methods. The investigation demonstrated that AS2-1 was a novel extracellular polysaccharide with different characterization from extracellular polysaccharides produced by other marine microorganisms.

  2. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

    Directory of Open Access Journals (Sweden)

    Daehwan Chung

    Full Text Available Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its

  3. Ribonuclease A mutant His(119)Asn : The role of histidine in catalysis

    NARCIS (Netherlands)

    Panov, Konstantin I.; Kolbanovskaya, Elena Yu.; Okorokov, Andrei L.; Panova, Tatiana B.; Terwisscha Van Scheltinga, Anke; Karpeisky, Marat Ya.; Beintema, Jaap J.

    1996-01-01

    Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies, The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates, Three amino acid residues located at the active site of RNase A (His(12), His(119), and

  4. Biofabrication of ZnS:Mn luminescent nanocrystals using histidine, hexahistidine, and His-tagged proteins: a comparison study.

    Science.gov (United States)

    Zhou, Weibin; Baneyx, François

    2014-08-15

    The ubiquitous hexahistidine purification tag has been used to conjugate proteins to the shell of CdSe:ZnS quantum dots (QDs) due to its affinity for surface-exposed Zn(2+) ions but little attention has been paid to the potential of His-tagged proteins for mineralizing luminescent ZnS nanocrystals. Here, we compare the ability of free histidine, a His tag peptide, His-tagged thioredoxin (TrxA, a monomeric protein), and N- and C-terminally His-tagged versions of Hsp31 (a homodimeric protein) to support the synthesis of Mn-doped ZnS nanocrystals from aqueous precursors under mild conditions of pH (8.2) and temperature (37°C). We find that: (1) it is possible to produce poor quality QDs when histidine is used at high (8 mM) concentration; (2) an increase in local histidine concentration through repetition of the amino acid as a His tag decreases the amount of needed reagent ≈10-fold and improves optical properties; (3) fusion of the same His tag to TrxA allows for ZnS:Mn QDs mineralization at micromolar concentrations; and (4) doubling the local hexahistidine concentration by exploiting Hsp31 dimerization further improves nanocrystal luminescence with the brightest particles obtained when His tags are spatially co-localized at the Hsp31 N-termini. Although hexahistidine tracts are not as efficient as combinatorially selected ZnS binding peptides at QD synthesis, it should be possible to use the large number of available His-tagged proteins and the synthesis approach described herein to produce luminescent nanoparticles whose protein shell carries a broad range of functions.

  5. Histidines in the octapeptide repeat of PrPC react with PrPSc at an acidic pH.

    Science.gov (United States)

    Cruite, Justin T; Abalos, Gil C; Bellon, Anne; Solforosi, Laura

    2011-03-15

    Cellular PrP is actively cycled between the cell surface and the endosomal pathway. The exact site and mechanism of conversion from PrP(C) to PrP(Sc) remain unknown. We have previously used recombinant antibodies containing grafts of PrP sequence to identify three regions of PrP(C) (aa23-27, 98-110, and 136-158) that react with PrP(Sc) at neutral pH. To determine if any regions of PrP(C) react with PrP(Sc) at an acidic pH similar to that of an endosomal compartment, we tested our panel of grafted antibodies for the ability to precipitate PrP(Sc) in a range of pH conditions. At pH near or lower than 6, PrP-grafted antibodies representing the octapeptide repeat react strongly with PrP(Sc) but not PrP(C). Modified grafts in which the histidines of the octarepeat were replaced with alanines did not react with PrP(Sc). PrP(Sc) precipitated by the octapeptide at pH 5.7 was able to seed conversion of normal PrP to PrP(Sc) in vitro. However, modified PrP containing histidine to alanine substitutions within the octapeptide repeats was still converted to PrP(Sc) in N2a cells. These results suggest that once PrP has entered the endosomal pathway, the acidic environment facilitates the binding of PrP(Sc) to the octarepeat of PrP(C) by the change in charge of the histidines within the octarepeat.

  6. Improving the binding capacity of Ni2+ decorated porous magnetic silica spheres for histidine-rich protein separation.

    Science.gov (United States)

    Benelmekki, M; Caparros, C; Xuriguera, E; Lanceros-Mendez, S; Rodriguez-Carmona, E; Mendoza, R; Corchero, J L; Martinez, Ll M

    2013-01-01

    Biomagnetic immobilization of histidine-rich proteins based on the single-step affinity adsorption of transition metal ions continues to be a suitable practice as a cost effective and a up scaled alternative to the to multiple-step chromatographic separations. In our previous work, we synthesised Porous Magnetic silica (PMS) spheres by one-step hydrothermal-assisted modified-stöber method. The obtained spheres were decorated with Ni(2+) and Co(2+), and evaluated for the capture of a H6-Tagged green fluorescence protein (GFP-H6) protein. The binding capacity of the obtained spheres was found to be slightly higher in the case Ni(2+) decorated PMS spheres (PMSNi). However, comparing with commercial products, the binding capacity was found to be lower than the expected. In this way, the present work is an attempt to improve the binding capacity of PMSNi to histidine-rich proteins. We find that increasing the amount of Ni(2+) onto the surface of the PMS spheres leads to an increment of the binding capacity to GFP-H6 by a factor of two. On the other hand, we explore how the size of histidine-rich protein can affect the binding capacity comparing the results of the GFP-6H to those of the His-tagged α-galactosidase (α-GLA). Finally, we demonstrate that the optimization of the magnetophoresis parameters during washing and eluting steps can lead to an additional improvement of the binding capacity. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis.

    Science.gov (United States)

    Sugimoto, S; Iwase, T; Sato, F; Tajima, A; Shinji, H; Mizunoe, Y

    2011-12-01

    Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression-secretion system. The esp gene was fused with the N-terminal Sec-dependent signal sequence of the B. choshinensis cell wall protein and a C-terminal hexa-histidine-tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20-ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1-l culture). Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  8. Pesticide residues in brain tissues of dairy cattle in Lembang

    Directory of Open Access Journals (Sweden)

    Indraningsih

    2006-03-01

    Full Text Available The use of pesticides to control plant diseases may cause residual formation in crops, its byproduct and environmental. Furthermore, the use of agriculture byproduct as animal feed may cause poisoning or residual formation in animal products. The purpose of this study is to investigate of pesticide residues in brain tissues of dairy cattle in relation to animal feed as a contamination source. Samples consisted of animal feeds (19 samples of fodder and 6 samples of feed, 31 samples of sera and 25 samples of brain tissues of dairy cattle collected from Lembang, West Java. Feeds and fodders were collected from dairy farms located in Lembang. Sera were directly collected from 31 heads of Frisien Holstein (FH cattle from the same location, while brain tissues of FH cattle were collected from a local animal slaughtering house. Pesticide residues were analysed using gas chromatography (GC. Both residues of organochlorines and organophosphates were detected from brain tissues with average residue concentration OP was 22.7 ppb and OC was 5.1 ppb and a total residue was 27.8 ppb. The pesticide residues in brain tissues are new information that should be taken into consideration since the Indonesian consumed this tissues as an oval. Although pesticides residue concentration was low, pathological changes were noted microscopically from the brain tissues including extracellular vacuolisation, focal necrosis, haemorrhages, dilatation of basement membrane without cellular infiltration. Both pesticide residues were also detected in sera, where OP (9.0 ppb was higher than OC (4.9 ppb. These pesticides were also detected in animal feeds consisting fodders and feeds. Residues of OP (12.0 ppb were higher than OC (1.8 ppb in feeds, but residues of OP (16.8 ppb were lower than OC (18.7 ppb in fodders. Although, pesticide residues in sera and brain tissues were below the maximum residue limits (MRL of fat, the presence of pesticides in brain tissues should be taken

  9. Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization

    Science.gov (United States)

    Vorontsov, Egor; Gallaud, Julien; Malosse, Christian; Michel, Valérie; Cavazza, Christine; Robbe-Saule, Marie; Richaud, Pierre; Chamot-Rooke, Julia; Brochier-Armanet, Céline; De Reuse, Hilde

    2015-01-01

    Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric

  10. Thermochemistry of alkali metal cation interactions with histidine: influence of the side chain.

    Science.gov (United States)

    Armentrout, P B; Citir, Murat; Chen, Yu; Rodgers, M T

    2012-12-06

    The interactions of alkali metal cations (M(+) = Na(+), K(+), Rb(+), Cs(+)) with the amino acid histidine (His) are examined in detail. Experimentally, bond energies are determined using threshold collision-induced dissociation of the M(+)(His) complexes with xenon in a guided ion beam tandem mass spectrometer. Analyses of the energy dependent cross sections provide 0 K bond energies of 2.31 ± 0.11, 1.70 ± 0.08, 1.42 ± 0.06, and 1.22 ± 0.06 eV for complexes of His with Na(+), K(+), Rb(+), and Cs(+), respectively. All bond dissociation energy (BDE) determinations include consideration of unimolecular decay rates, internal energy of reactant ions, and multiple ion-neutral collisions. These experimental results are compared to values obtained from quantum chemical calculations conducted previously at the MP2(full)/6-311+G(2d,2p), B3LYP/6-311+G(2d,2p), and B3P86/6-311+G(2d,2p) levels with geometries and zero point energies calculated at the B3LYP/6-311+G(d,p) level where Rb and Cs use the Hay-Wadt effective core potential and basis set augmented with additional polarization functions (HW*). Additional calculations using the def2-TZVPPD basis set with B3LYP geometries were conducted here at all three levels of theory. Either basis set yields similar results for Na(+)(His) and K(+)(His), which are in reasonable agreement with the experimental BDEs. For Rb(+)(His) and Cs(+)(His), the HW* basis set and ECP underestimate the experimental BDEs, whereas the def2-TZVPPD basis set yields results in good agreement. The effect of the imidazole side chain on the BDEs is examined by comparing the present results with previous thermochemistry for other amino acids. Both polarizability and the local dipole moment of the side chain are influential in the energetics.

  11. The histidine kinase AHK5 integrates endogenous and environmental signals in Arabidopsis guard cells.

    Directory of Open Access Journals (Sweden)

    Radhika Desikan

    2008-06-01

    Full Text Available Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H(2O(2. The mechanisms by which H(2O(2 integrates multiple signals via specific signalling pathways leading to stomatal closure is not known.Here, we identify a pathway by which H(2O(2, derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H(2O(2, which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H(2O(2, such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H(2O(2 production and H(2O(2 induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP flagellin, but not elf peptide, also exhibited reduced stomatal closure and H(2O(2 generation in ahk5 mutants.Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H(2O(2 homeostasis and is independent of ABA

  12. Improving the binding capacity of Ni2+ decorated porous magnetic silica spheres for histidine-rich protein separation

    OpenAIRE

    Benelmekki, Maria; Caparrós Vázquez, Cristina Maria; Xuriguera, Elena; Lanceros Méndez, Senentxu; Rodriguez-Carmona, Escar; Mendoza, R; Corchero, Jose Luis; Martinez, lluis Maria

    2012-01-01

    Biomagnetic immobilization of histidine-rich proteins based on the single-step affinity adsorption of transition metal ions continues to be a suitable practice as a cost effective and a up scaled alternative to the to multiple-step chromatographic separations. In our previous work [12], we synthesised Porous Magnetic silica (PMS) spheres by one-step hydrothermal-assisted modified-stöber method. The obtained spheres were decorated with Ni2+ and Co2+, and evaluated for the capture of a H6-Tagge...

  13. The complex extracellular biology of Streptomyces.

    Science.gov (United States)

    Chater, Keith F; Biró, Sandor; Lee, Kye Joon; Palmer, Tracy; Schrempf, Hildgund

    2010-03-01

    Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

  14. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

    Hromada, Carina; Mühleder, Severin; Grillari, Johannes; Redl, Heinz; Holnthoner, Wolfgang

    2017-01-01

    Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.

  15. Proteases decode the extracellular matrix cryptome.

    Science.gov (United States)

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Iron (III) porphyrin bearing 2,6-di-tert-butylphenol pendants deposited onto gold electrodes for amperometric determination of L-histidine.

    Science.gov (United States)

    Kurzatkowska, Katarzyna; Shpakovsky, Dmitry; Radecki, Jerzy; Radecka, Hanna; Jingwei, Zhang; Milaeva, Elena

    2009-04-15

    A sensitive amperometric sensor for determination of L-histidine was developed using gold electrode modified with Fe(III)-porphyrin bearing three 2,6-di-tert-butylphenol groups and one palmitoyl chain. Two methods of electrode modification were applied: direct chemisorption and embedment into dodecanethiol monolayer. Both types of electrodes were used for detection of L-histidine using Osteryoung square-wave voltammetry. The sensitivity of sensors presented towards L-histidine depends on the method of electrode modification. The detection limits observed for the electrodes incorporating with Fe(III)-porphyrin host by embedment and chemisorption were in 1 and 100 nM ranges, respectively. In addition, the determination of L-histidine with electrode modified by embedment technique was more precise, in comparison to that obtained by the direct chemisorption. Applicability of gold electrodes modified with Fe(III)-porphyrin for the direct electrochemical determination of L-histidine was demonstrated using the artificial matrix mimicking human serum.

  17. Phytoremediation of mixed-contaminated soil using the hyperaccumulator plant Alyssum lesbiacum: Evidence of histidine as a measure of phytoextractable nickel

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Andrew C. [Centre for Ecology and Hydrology-Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom)]. E-mail: acsi@ceh.ac.uk; Bell, Thomas [Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS (United Kingdom); Heywood, Chloe A. [Centre for Ecology and Hydrology-Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Smith, J.A.C. [Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB (United Kingdom); Thompson, Ian P. [Centre for Ecology and Hydrology-Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom)

    2007-05-15

    In this study we examine the effects of polycyclic aromatic hydrocarbons (PAHs) on the ability of the hyperaccumulator plant Alyssum lesbiacum to phytoextract nickel from co-contaminated soil. Planted and unplanted mesocosms containing the contaminated soils were repeatedly amended with sorbitan trioleate, salicylic acid and histidine in various combinations to enhance the degradation of two PAHs (phenanthrene and chrysene) and increase nickel phytoextraction. Plant growth was negatively affected by PAHs; however, there was no significant effect on the phytoextraction of Ni per unit biomass of shoot. Exogenous histidine did not increase nickel phytoextraction, but the histidine-extractable fraction of soil nickel showed a high correlation with phytoextractable nickel. These results indicate that Alyssum lesbiacum might be effective in phytoextracting nickel from marginally PAH-contaminated soils. In addition, we provide evidence for the broader applicability of histidine for quantifying and predicting Ni phytoavailability in soils. - Alyssum lesbiacum was shown to phytoextract nickel from PAH-contaminated soils from which the pool of nickel accessed for phytoextraction is closely modelled by a histidine-soil extract.

  18. Sharing Residual Liability

    DEFF Research Database (Denmark)

    Carbonara, Emanuela; Guerra, Alice; Parisi, Francesco

    2016-01-01

    Economic models of tort law evaluate the efficiency of liability rules in terms of care and activity levels. A liability regime is optimal when it creates incentives to maximize the value of risky activities net of accident and precaution costs. The allocation of primary and residual liability...... allows policy makers to induce parties to undertake socially desirable care and activity levels. Traditionally, tort law systems have assigned residual liability either entirely on the tortfeasor or entirely on the victim. In this paper, we unpack the cheapest-cost-avoider principle to consider...

  19. Residual-stress measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ezeilo, A.N.; Webster, G.A. [Imperial College, London (United Kingdom); Webster, P.J. [Salford Univ. (United Kingdom)

    1997-04-01

    Because neutrons can penetrate distances of up to 50 mm in most engineering materials, this makes them unique for establishing residual-stress distributions non-destructively. D1A is particularly suited for through-surface measurements as it does not suffer from instrumental surface aberrations commonly found on multidetector instruments, while D20 is best for fast internal-strain scanning. Two examples for residual-stress measurements in a shot-peened material, and in a weld are presented to demonstrate the attractive features of both instruments. (author).

  20. The dapE-encoded N-succinyl-L,L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

    OpenAIRE

    Gillner, Danuta M.; Bienvenue, David L.; Nocek, Boguslaw P; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C.

    2008-01-01

    The catalytic and structural properties of the H67A and H349A altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from H. influenzae were investigated. Based on sequence alignment with CPG2 both H67 and H349 were predicted to be Zn(II) ligands. Catalytic activity was observed for the H67A altered DapE enzyme which exhibited kcat = 1.5 ± 0.5 sec−1 and Km = 1.4 ± 0.3 mM. No catalytic activity was observed for H349A under the experimental conditions used. The EPR and ele...

  1. Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, H; Stover, AG; Driks, A; Venema, G; Bron, S; van Dijl, JM

    2000-01-01

    Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally

  2. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  3. Analysis of the inter- and extracellular formation of platinum nanoparticles by Fusarium oxysporum f. sp. lycopersici using response surface methodology

    Energy Technology Data Exchange (ETDEWEB)

    Riddin, T L [Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, PO Box 94, Grahamstown (South Africa); Gericke, M [MINTEK, Private Bag X3015, Randburg 2125 (South Africa); Whiteley, C G [Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, PO Box 94, Grahamstown (South Africa)

    2006-07-28

    Fusarium oxysporum fungal strain was screened and found to be successful for the inter- and extracellular production of platinum nanoparticles. Nanoparticle formation was visually observed, over time, by the colour of the extracellular solution and/or the fungal biomass turning from yellow to dark brown, and their concentration was determined from the amount of residual hexachloroplatinic acid measured from a standard curve at 456 nm. The extracellular nanoparticles were characterized by transmission electron microscopy. Nanoparticles of varying size (10-100 nm) and shape (hexagons, pentagons, circles, squares, rectangles) were produced at both extracellular and intercellular levels by the Fusarium oxysporum. The particles precipitate out of solution and bioaccumulate by nucleation either intercellularly, on the cell wall/membrane, or extracellularly in the surrounding medium. The importance of pH, temperature and hexachloroplatinic acid (H{sub 2}PtCl{sub 6}) concentration in nanoparticle formation was examined through the use of a statistical response surface methodology. Only the extracellular production of nanoparticles proved to be statistically significant, with a concentration yield of 4.85 mg l{sup -1} estimated by a first-order regression model. From a second-order polynomial regression, the predicted yield of nanoparticles increased to 5.66 mg l{sup -1} and, after a backward step, regression gave a final model with a yield of 6.59 mg l{sup -1}.

  4. Analysis of the inter- and extracellular formation of platinum nanoparticles by Fusarium oxysporum f. sp. lycopersici using response surface methodology

    Science.gov (United States)

    Riddin, T. L.; Gericke, M.; Whiteley, C. G.

    2006-07-01

    Fusarium oxysporum fungal strain was screened and found to be successful for the inter- and extracellular production of platinum nanoparticles. Nanoparticle formation was visually observed, over time, by the colour of the extracellular solution and/or the fungal biomass turning from yellow to dark brown, and their concentration was determined from the amount of residual hexachloroplatinic acid measured from a standard curve at 456 nm. The extracellular nanoparticles were characterized by transmission electron microscopy. Nanoparticles of varying size (10-100 nm) and shape (hexagons, pentagons, circles, squares, rectangles) were produced at both extracellular and intercellular levels by the Fusarium oxysporum. The particles precipitate out of solution and bioaccumulate by nucleation either intercellularly, on the cell wall/membrane, or extracellularly in the surrounding medium. The importance of pH, temperature and hexachloroplatinic acid (H2PtCl6) concentration in nanoparticle formation was examined through the use of a statistical response surface methodology. Only the extracellular production of nanoparticles proved to be statistically significant, with a concentration yield of 4.85 mg l-1 estimated by a first-order regression model. From a second-order polynomial regression, the predicted yield of nanoparticles increased to 5.66 mg l-1 and, after a backward step, regression gave a final model with a yield of 6.59 mg l-1.

  5. Designing with residual materials

    NARCIS (Netherlands)

    Walhout, W.; Wever, R.; Blom, E.; Addink-Dölle, L.; Tempelman, E.

    2013-01-01

    Many entrepreneurial businesses have attempted to create value based on the residual material streams of third parties. Based on ‘waste’ materials they designed products, around which they built their company. Such activities have the potential to yield sustainable products. Many of such companies

  6. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-27

    This report compared the composition of samples from Wesseling and Leuna. In each case the sample was a residue from carbonization of the residues from hydrogenation of the brown coal processed at the plant. The composition was given in terms of volatile components, fixed carbon, ash, water, carbon, hydrogen, oxygen, nitrogen, volatile sulfur, and total sulfur. The result of carbonization was given in terms of (ash and) coke, tar, water, gas and losses, and bitumen. The composition of the ash was given in terms of silicon dioxide, ferric oxide, aluminum oxide, calcium oxide, magnesium oxide, potassium and sodium oxides, sulfur trioxide, phosphorus pentoxide, chlorine, and titanium oxide. The most important difference between the properties of the two samples was that the residue from Wesseling only contained 4% oil, whereas that from Leuna had about 26% oil. Taking into account the total amount of residue processed yearly, the report noted that better carbonization at Leuna could save 20,000 metric tons/year of oil. Some other comparisons of data included about 33% volatiles at Leuna vs. about 22% at Wesseling, about 5 1/2% sulfur at Leuna vs. about 6 1/2% at Leuna, but about 57% ash for both. Composition of the ash differed quite a bit between the two. 1 table.

  7. Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases.

    Science.gov (United States)

    Shan, Ling; Hofman, Michel A; van Wamelen, Daniel J; Van Someren, Eus J W; Bao, Ai-Min; Swaab Dick, F

    2012-05-01

    Neuronal histamine shows diurnal rhythms in rodents and plays a major role in the maintenance of vigilance. No data are available on its diurnal fluctuation in humans, either in health or in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD), or Huntington disease (HD), all of which are characterized by sleep-wake disturbances. Quantitative in situ hybridization was used to study the mRNA expression of histidine decarboxylase (HDC), the key enzyme of histamine production in the tuberomammillary nucleus (TMN) in postmortem human hypothalamic tissue, obtained from 33 controls and 31 patients with a neurodegenerative disease-PD (n = 15), AD (n = 9), and HD (n = 8)-and covering the full 24-h cycle with respect to clock time of death. HDC-mRNA levels in controls were found to be significantly higher during the daytime than at night (e.g., 08:01-20:00 versus 20:01-08:00, P = 0.004). This day-night fluctuation was markedly different in patients with neurodegenerative diseases. The diurnal fluctuation of HDC-mRNA expression in human TMN supports a role for neuronal histamine in regulating day-night rhythms. Future studies should investigate histamine rhythm abnormalities in neurodegenerative disorders. Shan L; Hofman MA; van Wamelen DJ; Van Someren EJW; Bao AM; Swaab DF. Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases.

  8. Mechanistic insights revealed by the crystal structure of a histidine kinase with signal transducer and sensor domains.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available Two-component systems (TCSs are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK and a response regulator (RR, which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS, DHp, and catalytic and ATP binding domain (CA. The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.

  9. UmTco1, a Hybrid Histidine Kinase Gene, Is Essential for the Sexual Development and Virulence of Ustilago maydis.

    Science.gov (United States)

    Yun, Yeo Hong; Oh, Man Hwan; Kim, Jun Young; Kim, Seong Hwan

    2017-05-28

    Hybrid histidine kinase is part of a two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. The Tco1 gene in human pathogen Cryptococcus neoformans encodes a hybrid histidine kinase and is important for pathogenesis. In this study, we identified a Tco1 homolog, UmTco1, in the maize pathogen Ustilago maydis by bioinformatics analysis. To explore the role of UmTco1 in the survival of U. maydis under environmental stresses and its pathogenesis, Δumtco1 mutants were constructed by allelic exchange. The growth of Δumtco1 mutants was significantly impaired when they were cultured under hyperosmotic stress. The Δumtco1 mutants exhibited increased resistance to antifungal agent fludioxonil. In particular, the Δumtco1 mutants were unable to produce cytokinesis or conjugation tubes, and to develop fuzzy filaments, resulting in impaired mating between compatible strains. The expression levels of Prf1, Pra1, and Mfa1, which are involved in the pheromone pathway, were significantly decreased in the Δumtco1 mutants. In inoculation tests to the host plant, the Δumtco1 mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that UmTco1 plays important roles in the survival under hyperosmotic stress, and contributes to cytokinesis, sexual development, and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.

  10. The nonoxidative conversion of nitroethane to ethylnitronate in Neurospora crassa 2-nitropropane dioxygenase is catalyzed by histidine 196.

    Science.gov (United States)

    Francis, Kevin; Gadda, Giovanni

    2008-09-02

    The deprotonation of nitroethane catalyzed by Neurospora crassa 2-nitropropane dioxygenase was investigated by measuring the formation and release of ethylnitronate formed in turnover as a function of pH and through mutagenesis studies. Progress curves for the enzymatic reaction obtained by following the increase in absorbance at 228 nm over time were visibly nonlinear, requiring a logarithmic approximation of the initial reaction rates for the determination of the kinetic parameters of the enzyme. The pH dependence of the second-order rate constant k cat/ K m with nitroethane as substrate implicates the presence of a group with a p K a of 8.1 +/- 0.1 that must be unprotonated for nitronate formation. Mutagenesis studies suggest that this group is histidine 196 as evident from the inability of a H196N variant form of the enzyme to catalyze the formation of ethylnitronate from nitroethane. Replacement of histidine 196 with asparagine resulted in an approximately 15-fold increase in the k cat/ K m with ethylnitronate as compared to the wild-type, which results from the inability of the mutant enzyme to undergo nonoxidative turnover. The results presented herein are consistent with a branched catalytic mechanism for the enzyme in which the ethylnitronate intermediate formed from the H196-catalyzed deprotonation of nitroethane partitions between release from the active site and oxidative denitrification to yield acetaldehyde and nitrite.

  11. Antioxidation status and histidine dipeptides content in broiler blood and muscles depending on protein sources in feed.

    Science.gov (United States)

    Kopeć, W; Jamroz, D; Wiliczkiewicz, A; Biazik, E; Hikawczuk, T; Skiba, T; Pudło, A; Orda, J

    2013-06-01

    One-day-old chickens were fed mixtures containing different raw materials (fish by-products meal, porcine blood cells meal, blood meal, wheat gluten, fodder yeast), as a source of histidine and β-alanine - components of carnosine. Control birds were administered a feed mixture, in which soy bean meal was the main protein source. The bodyweight, feed consumption and conversion, antioxidant characteristics and histidine dipeptides content in blood and muscles, and also amino acid composition of chicken meat on day 34 post-hatch were recorded. The best (p chickens fed mixture containing porcine blood cells meal. In blood plasma of control chickens, a significantly (p chickens fed mixtures with blood by-products. Insignificant differences in both carnosine and anserine levels in plasma between treatments were noted. Breast muscles from control birds were characterized by lower activity of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) (p chickens fed blood by-products. Improved ability to reduce ferric ions (FRAP) (p content in meat from chickens fed blood cell meal were recorded. No direct relations between amino acids content in feed mixtures and in meat were observed. © 2012 Blackwell Verlag GmbH.

  12. Isolation and transcript analysis of two-component histidine kinase gene Le.nik1 in Shiitake mushroom, Lentinula edodes.

    Science.gov (United States)

    Szeto, Carol Y Y; Wong, Queenie W L; Leung, Grace S; Kwan, H S

    2008-01-01

    Le.nik1, a two-component histidine kinase gene of Lentinula edodes, the Shiitake mushroom, was identified. The relationship between this two-component signal transduction system and mushroom development was studied. We used a modified RNA arbitrarily-primed PCR (RAP-PCR) method to isolate Le.nik1 as a differentially expressed gene during L. edodes development. We determined the 6.29kb full-length cDNA sequence of Le.nik1. It had high sequence homology to Neurospora crassa nik1, which encoded a histidine kinase essential for development and osmotic response. In L. edodes, the expression level of Le.nik1 was highest during primordium formation and fruiting body maturation. The transcripts were localized predominantly in the developing hymenophores, or mushroom gills, which may indicate the role of a two-component signal transduction system in cell differentiation during mushroom development. Mannitol stress influenced transcript expression of Le.nik1, suggesting that it may be involved in osmo-sensing and regulation. To our knowledge, this is the first report on the two-component system in mushrooms and the first analysis on the distribution of Le.nik1 transcript in the course of fruiting body formation and in parts of fruiting bodies.

  13. Optimization of extracellular catalase production from Aspergillus ...

    African Journals Online (AJOL)

    The studies of the effect of each variable and the establishment of a correlation between the response of enzyme activity and variables revealed that the link is a multiple linear regression form. The optimization was carried out through a simplex algorithm. The amount of extracellular catalase produced by the strain in the ...

  14. Optimization of extracellular polysaccharide production in ...

    African Journals Online (AJOL)

    sunny

    2014-11-26

    Nov 26, 2014 ... The present study was conducted to optimize the media composition through response surface methodology (RSM) for extracellular polysaccharide (EPS) production in Halobacillus trueperi AJSK strain isolated from the salt pan. Halobacillus trueperi was i d e n t i f i e d with morphological, biochemical ...

  15. Optimization of extracellular polysaccharide production in ...

    African Journals Online (AJOL)

    The present study was conducted to optimize the media composition through response surface methodology (RSM) for extracellular polysaccharide (EPS) production in Halobacillus trueperi AJSK strain isolated from the salt pan. Halobacillus trueperi was identified with morphological, biochemical characteristics as well as ...

  16. Preliminary research of recombinant matrix extracellular ...

    African Journals Online (AJOL)

    ... and predentin, but not by dental pulp cells. Furthermore, we used von kossa staining and the results suggested that, MEPE could induce mineralization and we propose that this protein had a potential effect on dental rehabilitation. Key words: Matrix extracellular phosphoglycoprotein (MEPE), mineralization Von kossa.

  17. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  18. Interaction of acetamiprid with extracellular polymeric substances ...

    African Journals Online (AJOL)

    Extracellular polymeric substances (EPS) are important components of activated sludge and it plays an important role in removing pollutants. The interaction between EPS and organic pollutants is still little known. In the present study, the interaction of soluble/bound EPS with acetamiprid, a neonicotinoid insecticide, was ...

  19. Production of extracellular aspartic protease in submerged ...

    African Journals Online (AJOL)

    Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h ...

  20. Towards traceable size determination of extracellular vesicles

    NARCIS (Netherlands)

    Varga, Zoltán; Yuana, Yuana; Grootemaat, Anita E.; van der Pol, Edwin; Gollwitzer, Christian; Krumrey, Michael; Nieuwland, Rienk

    2014-01-01

    Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. In this manuscript, the size distribution of an

  1. Extracellular matrix and tissue engineering applications

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Moroni, Lorenzo; van Blitterswijk, Clemens; de Boer, Jan

    2009-01-01

    The extracellular matrix is a key component during regeneration and maintenance of tissues and organs, and it therefore plays a critical role in successful tissue engineering as well. Tissue engineers should recognise that engineering technology can be deduced from natural repair processes. Due to

  2. Characterization of Extracellular Vesicles using Raman Spectroscopy

    NARCIS (Netherlands)

    Lee, Wooje; Nanou, Afroditi; Terstappen, Leonardus Wendelinus Mathias Marie; Rho, Hoon Suk; le Gac, Severine; Offerhaus, Herman L.

    2017-01-01

    In this research, we aim to characterize extracellular vesicles(EVs) with Confocal Raman spectroscopy to reveal relevant spectral lines that signify differences between EVs derived from different cell lines. In the first stage we performed confocal Raman measurements on various EV samples. For these

  3. Assessment of extracellular dehydration using saliva osmolality.

    Science.gov (United States)

    Ely, Brett R; Cheuvront, Samuel N; Kenefick, Robert W; Spitz, Marissa G; Heavens, Kristen R; Walsh, Neil P; Sawka, Michael N

    2014-01-01

    When substantial solute losses accompany body water an isotonic hypovolemia (extracellular dehydration) results. The potential for using blood or urine to assess extracellular dehydration is generally poor, but saliva is not a simple ultra-filtrate of plasma and the autonomic regulation of salivary gland function suggests the possibility that saliva osmolality (Sosm) may afford detection of extracellular dehydration via the influence of volume-mediated factors. This study aimed to evaluate the assessment of extracellular dehydration using Sosm. In addition, two common saliva collection methods and their effects on Sosm were compared. Blood, urine, and saliva samples were collected in 24 healthy volunteers during paired euhydration and dehydration trials. Furosemide administration and 12 h fluid restriction were used to produce extracellular dehydration. Expectoration and salivette collection methods were compared in a separate group of eight euhydrated volunteers. All comparisons were made using paired t-tests. The diagnostic potential of body fluids was additionally evaluated. Dehydration (3.1 ± 0.5% loss of body mass) decreased PV (-0.49 ± 0.12 L; -15.12 ± 3.94% change), but Sosm changes were marginal (diagnostic accuracy was poor (AUC = 0.77-0.78) for all body fluids evaluated. Strong agreement was observed between Sosm methods (Expectoration: 61 ± 10 mmol/kg, Salivette: 61 ± 8 mmol/kg, p > 0.05). Extracelluar dehydration was not detectable using plasma, urine, or saliva measures. Salivette and expectoration sampling methods produced similar, consistent results for Sosm, suggesting no methodological influence on Sosm.

  4. Cysteine cathepsins and extracellular matrix degradation.

    Science.gov (United States)

    Fonović, Marko; Turk, Boris

    2014-08-01

    Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins. In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis. Cysteine cathepsins are among the major proteases involved in ECM remodeling, and their role is not limited to degradation only. Deregulation of their activity is linked with numerous ECM-linked diseases and they are now validated targets in a number of them. Cathepsins S and K are the most attractive targets, especially cathepsin K as a major therapeutic target for osteoporosis with drugs targeting it in advanced clinical trials. Due to their major role in ECM remodeling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All

  5. Residual contaminants in milk

    Directory of Open Access Journals (Sweden)

    Nevijo Zdolec

    2006-07-01

    Full Text Available Various chemical agents are used during the whole production chain of milk and dairy products. Production of feedingstuffs is accompanied with pesticide usage, which may remain in environment, thus are transported through feeding into animals, animal products and finally in human organism. Preparation procedure and storage conditions of feed also influence on milk safety in the sense of mycotoxins entering into the food chain. Chemical agents are, on daily basis, used on dairy farms either as detergents or disinfections. The residuals of cleaning agents might remain in milk if the cleaning agents and its dosage are not performed adequately. Besides already mentioned agents, a great influence in milk production can bee seen through veterinary drugs usage, particularly antibacterial drugs (mastitis. Proper application of drugs and by following legal recommendation, a by-reactions can be avoided such as allergic reaction in humans, development of resisting bacteria or even undesirable influence on starter cultures in dairy products manufacture. The maximum residue limits, monitoring plan as well as sampling procedures are set up within the harmonization of Croatian and European legislation, in order to provide official control of residues in foodstuffs of animal origin.

  6. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-30

    This report gave a record of the composition of several samples of residues from carbonization of various hydrogenation residue from processing some type of coal or tar in the Bergius process. These included Silesian bituminous coal processed at 600 atm. with iron catalyst, in one case to produce gasoline and middle oil and in another case to produce heavy oil excess, Scholven coal processed at 250 atm. with tin oxalate and chlorine catalyst, Bruex tar processed in a 10-liter oven using iron catalyst, and a pitch mixture from Welheim processed in a 10-liter over using iron catalyst. The values gathered were compared with a few corresponding values estimated for Boehlen tar and Gelsenberg coal based on several assumptions outlined in the report. The data recorded included percentage of ash in the dry residue and percentage of carbon, hydrogen, oxygen, nitrogen, chlorine, total sulfur, and volatile sulfur. The percentage of ash varied from 21.43% in the case of Bruex tar to 53.15% in the case of one of the Silesian coals. Percentage of carbon varied from 44.0% in the case of Scholven coal to 78.03% in the case of Bruex tar. Percentage of total sulfur varied from 2.28% for Bruex tar to a recorded 5.65% for one of the Silesian coals and an estimated 6% for Boehlen tar. 1 table.

  7. Structure of the Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme and enzymatic inactivation by mutation of the heme coordinating residue His-193

    Energy Technology Data Exchange (ETDEWEB)

    Suits,M.; Jaffer, N.; Jia, Z.

    2006-01-01

    Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 {angstrom} resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a {beta}-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain, assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the {alpha}-meso carbon position where O{sub 2} is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.

  8. Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1*

    Science.gov (United States)

    Hong, Tae-Joon; Kim, Sangkyu; Wi, Ah Ram; Lee, Peter; Kang, Miae; Jeong, Jae-Hoon; Hahn, Ji-Sook

    2013-01-01

    Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca2+, which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca2+. Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones. PMID:23184943

  9. Involvement of extracellular matrix constituents in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  10. Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

    DEFF Research Database (Denmark)

    Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern

    2012-01-01

    Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional....... Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...

  11. Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

    Directory of Open Access Journals (Sweden)

    Jessica Capraro

    Full Text Available The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

  12. Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

    Science.gov (United States)

    Capraro, Jessica; Sessa, Fabio; Magni, Chiara; Scarafoni, Alessio; Maffioli, Elisa; Tedeschi, Gabriella; Croy, Ron R D; Duranti, Marcello

    2015-01-01

    The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

  13. Use of histidine dipeptides and myoglobin to monitor adulteration of cooked beef with meat from other species.

    Science.gov (United States)

    Carnegie, P R; Ilic, M Z; Etheridge, M O; Stuart, S

    1985-08-01

    A new high performance liquid chromatography (HPLC) method was used to monitor the adulteration of cooked beef products with meat from other species. The ratio of the histidine dipeptides anserine and carnosine which are present in skeletal muscle, are so different between sheep, cattle, horse and kangaroo that detection of adulteration can be rapidly achieved by chromatography on a Partisil-10 SCX column with 0.2 M lithium formate, pH 2.9. To obtain a definitive identification of the adulterant it was necessary to also examine the electrophoretic mobility of myoglobin in sodium dodecylsulphate gels. One brand of "beefsteak" pie was found to actually be a mixture of mutton and beef.

  14. Potentiometric determination of the dissociation constants of L-histidine, proline and tryptophane in various hydroorganic media.

    Science.gov (United States)

    Azab, H A; El-Nady, A M; El-Shatoury, S A; Hassan, A

    1994-08-01

    The dissociation constant values of L-histidine, proline and tryptophane were determined at 25 +/- 0.1 degrees C by potentiometric pH titration in pure water and different hydroorganic solvent media. The organic solvents used were methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, acetone and dioxane. Initial estimates of the dissociation constant values of the different amino acids studied have been refined with ESAP2M computer program. It was observed that changing the medium permittivity as the solvent is enriched in methanol or ethanol has little influence on the pK*(a) values of the amino acids studied. The results obtained are discussed in terms of average macroscopic properties of the mixed solvents and the possible variation in microheterogeneity of the salvation shells around the solute.

  15. Plasmodium falciparum histidine-rich protein II causes vascular leakage and exacerbates experimental cerebral malaria in mice.

    Science.gov (United States)

    Pal, Priya; Balaban, Amanda E; Diamond, Michael S; Sinnis, Photini; Klein, Robyn S; Goldberg, Daniel E

    2017-01-01

    A devastating complication of Plasmodium falciparum infection is cerebral malaria, in which vascular leakage and cerebral swelling lead to coma and often death. P. falciparum produces a protein called histidine-rich protein II (HRPII) that accumulates to high levels in the bloodstream of patients and serves as a diagnostic and prognostic marker for falciparum malaria. Using a human cerebral microvascular endothelial barrier model, we previously found that HRPII activates the endothelial cell inflammasome, resulting in decreased integrity of tight junctions and increased endothelial barrier permeability. Here, we report that intravenous administration of HRPII induced blood-brain barrier leakage in uninfected mice. Furthermore, HRPII infusion in P. berghei-infected mice increased early mortality from experimental cerebral malaria. These data support the hypothesis that HRPII is a virulence factor that contributes to cerebral malaria by compromising the integrity of the blood-brain barrier.

  16. Synthesis of poly(methyl methacrylate)-block-poly(L-histidine) and its use as a hybrid silver nanoparticle conjugate.

    Science.gov (United States)

    Shin, Nam Ho; Lee, Jin Kyu; Li, Haiqing; Ha, Chang-Sik; Shchipunov, Yury A; Kim, Il

    2010-10-01

    Poly[(methyl methacrylate)-block-poly(L-histidine)] (PMMA-b-PHIS) was synthesized by combining atom transfer radical polymerization and living ring-opening polymerization of alpha-amino acid-N-carboxyanhydride. The resulting hybrid block copolymer forms reverse micelles in the mixture solution of water and N,N-dimethylformamide (DMF) and self-assembles into PHIS/PMMA core/shell spheres with controllable size in the range of 80 to 250 nm depending on the micellization temperature. The self-assembly of PMMA-b-PHIS was carried out in H2O/DMF (3/7) mixture in the presence of AgNO3. Reduction of the resulting Ag ions encapsulated inside of the reverse micelles yielded an attractive Ag nanoparticle core/polymer shell conjugate system.

  17. Assembly of the transmembrane domain of E. coli PhoQ histidine kinase: implications for signal transduction from molecular simulations.

    Directory of Open Access Journals (Sweden)

    Thomas Lemmin

    Full Text Available The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.

  18. Arabidopsis histidine-containing phosphotransfer factor 4 (AHP4) negatively regulates secondary wall thickening of the anther endothecium during flowering.

    Science.gov (United States)

    Jung, Kwang Wook; Oh, Seung-Ick; Kim, Yun Young; Yoo, Kyoung Shin; Cui, Mei Hua; Shin, Jeong Sheop

    2008-04-30

    Cytokinins are essential hormones in plant development. Arabidopsis histidine-containing phosphotransfer proteins (AHPs) are mediators in a multistep phosphorelay pathway for cytokinin signaling. The exact role of AHP4 has not been elucidated. In this study, we demonstrated young flower-specific expression of AHP4, and compared AHP4-overexpressing (Ox) trangenic Arabidopsis lines and an ahp4 knock-out line. AHP4-Ox plants had reduced fertility due to a lack of secondary cell wall thickening in the anther endothecium and inhibition of IRREGURAR XYLEMs (IRXs) expression in young flowers. Conversely, ahp4 anthers had more lignified anther walls than the wild type, and increased IRXs expression. Our study indicates that AHP4 negatively regulates thickening of the secondary cell wall of the anther endothecium, and provides new insight into the role of cytokinins in formation of secondary cell walls via the action of AHP4.

  19. Growth, structural and optical characterization of L-histidine 4-nitrophenolate (LHPNP) single crystals for NLO applications

    Energy Technology Data Exchange (ETDEWEB)

    Mahadevan, M., E-mail: devanphysics@gmail.com [Department of Physics, Adhiparasakthi Engineering College, Melmaruvathur - 603319 (India); Ramachandran, K., E-mail: ramach76@yahoo.com [Department of Physics, SRM University - Vadapalani Campus, Chennai -600026 (India); Anandan, P., E-mail: anandantcet@gmail.com [Department of Physics, Thiruvalluvar College of Engineering and Technology, Vandavasi-604 505, India and Research Institute of Electronics, Shizuoka University, 3-5-1 Johoku, Naka-Ku, Hamamatsu 432-8011 (Japan); Arivanandhan, M., E-mail: arivucz@gmail.com, E-mail: royhaya@ipc.shizuoka.ac.jp; Hayakawa, Y., E-mail: arivucz@gmail.com, E-mail: royhaya@ipc.shizuoka.ac.jp [Research Institute of Electronics, Shizuoka University, 3-5-1 Johoku, Naka-Ku, Hamamatsu 432-8011 (Japan)

    2014-10-15

    Using slow evaporation solution growth technique, single crystals of L-histidine-4-nitro phenolate has been grown from the solution. Structural analyses were carried out by powder x-ray diffraction, FT-Raman, Fourier Transform Infrared and Nuclear Magnetic Resonance spectral methods to conform the grown crystals. Thermal stability of the grown crystals was studied by thermo-gravimetric (TG) and differential thermal analyses (DTA). UV-Vis spectral analysis has been carried out to find the transparency of the grown crystal. Nonlinear optical property has been confirmed by Kurtz powder technique. The PL measurements were carried out in Perkin Elmer LS 55 Luminescence spectrometer using 410 nm as excitation wavelength. The observed properties have confirmed that the grown crystal is suitable for nonlinear optical applications.

  20. Structural and functional analogies and differences between histidine decarboxylase and aromatic l-amino acid decarboxylase molecular networks: Biomedical implications.

    Science.gov (United States)

    Sanchez-Jiménez, Francisca; Pino-Ángeles, Almudena; Rodríguez-López, Rocio; Morales, María; Urdiales, José Luis

    2016-12-01

    Human histidine decarboxylase (HDC) and dopa decarboxilase (DDC) are highly homologous enzymes responsible for the synthesis of biogenic amines (BA) like histamine, and serotonin and dopamine, respectively. The enzymes share many structural and functional analogies, while their product metabolisms also follow similar patterns that are confluent in some metabolic steps. They are involved in common physiological functions, such as neurotransmission, gastrointestinal track function, immunity, cell growth and cell differentiation. As a consequence, metabolic elements of both BA subfamilies are also co-participants in a long list of human diseases. This review summarizes the analogies and differences in their origin (HDC and DDC) as well as their common pathophysiological scenarios. The major gaps of information are also underlined, as they delay the possibility of holistic approaches that would help personalized medicine and pharmacological initiatives for prevalent and rare diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Detection of Cu2+ in Water Based on Histidine-Gold Labeled Multiwalled Carbon Nanotube Electrochemical Sensor

    Directory of Open Access Journals (Sweden)

    Rilong Zhu

    2017-01-01

    Full Text Available Based on the strong interaction between histidine and copper ions and the signal enhancement effect of gold-labeling carbon nanotubes, an electrochemical sensor is established and used to measure copper ions in river water. In this study the results show that the concentrations of copper ion have well linear relationship with the peak current in the range of 10−11–10−7 mol/L, and the limit of detection is 10−12 mol/L. When using this method to detect copper ions in the Xiangjiang River, the test results are consistent with the atomic absorption method. This study shows that the sensor is convenient to be used in daily monitoring of copper ions in river water.

  2. Hg-coordination studies of oligopeptides containing cysteine, histidine and tyrosine by $^{199m}$Hg-TDPAC

    CERN Document Server

    Ctortecka, B; Mallion, S; Butz, T; Hoffmann, R

    1999-01-01

    In order to study the interaction of histidine- and tyrosine- containing peptide chains with Hg(II), the nuclear quadrupole interaction (NQI) of /sup 199m/Hg in the Hg complexes of the oligopeptides alanyl-alanyl-histidyl-alanyl-alanine-amid (AAHAA-NH /sub 2/) and alanyl-alanyl-tyrosyl-alanyl-alanine-amid (AAYAA-NH/sub 2/) was determined by time differential perturbed angular correlation and is compared with previous data on alanyl-alanyl-cysteyl-alanyl- alanyl (AACAA-OH). The /sup 199m/Hg-NQIs depend on the oligopeptide to Hg(II) stoichiometry and indicate that two-fold and four-fold coordinations occur for the bound Hg(II). (12 refs).

  3. DETERMINATION OF THE 3-DIMENSIONAL SOLUTION STRUCTURE OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA-COLI USING MULTIDIMENSIONAL NMR-SPECTROSCOPY

    NARCIS (Netherlands)

    VANNULAND, NAJ; GROTZINGER, J; DIJKSTRA, K; SCHEEK, RM; ROBILLARD, GT

    1992-01-01

    We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on N-15-enriched and C-13/N-15-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. &

  4. The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase/Peptidase Domain

    Science.gov (United States)

    Donovan, David M.; Foster-Frey, Juli; Dong, Shengli; Rousseau, Geneviève M.; Moineau, Sylvain; Pritchard, David G.

    2006-01-01

    The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity. PMID:16820517

  5. Examination of Correlation between Histidine and Cadmium Absorption by Eleagnus angustifolia L., Vitis vinifera L. and Nerium oleander L. Using HPLC-MS and ICP-MS.

    Science.gov (United States)

    Ozen, Sukran Akkus; Yaman, Mehmet

    2016-02-01

    In this study, HPLC-MS and ICP-MS methods wereused for the determination of histidine and cadmium in Eleagnus angustifolia L., Vitis vinifera L. and Nerium oleander L. leaves taken from industrial area including Gaziantep and Bursa cities. To histidine determination by HPLC-MS, flow rate of mobile phase, fragmentor potential, injection volume and column temperature were optimized as 0.2 mL · min⁻¹, 70 V, 15 µL and 20 °C, respectively. For extraction of histidine from plants, distilled water was used by applying on 90 °C and 30 min. The concentrations (as mg · kg⁻¹) of histidine were found to be in range of 8~22 for Eleagnus angustifolia L., 10~33 for Vitis vinifera L. and 6~11 for Nerium oleander L. The concentrations of cadmium were found to be in ranges of 6~21 µg · kg⁻¹ for Vitis vinifera L. 15~110 µg · kg⁻¹ for Eleagnus angustifolia L. and 63~218 µg · kg⁻¹ for Nerium oleander L.

  6. Contribution of Histidine and Lysine to the Generation of Volatile Compounds in Jinhua Ham Exposed to Ripening Conditions Via Maillard Reaction.

    Science.gov (United States)

    Zhu, Chao-Zhi; Zhao, Jing-Li; Tian, Wei; Liu, Yan-Xia; Li, Miao-Yun; Zhao, Gai-Ming

    2017-12-01

    To evaluate the role of Maillard reactions in the generation of flavor compounds in Jinhua ham, the reactions of glucose and ethanal with histidine and lysine, respectively, were studied by simulating the ripening conditions of Jinhua ham. The volatile products produced were analyzed using solid phase microextraction-gas chromatography/mass spectrometry. The results showed that 8 volatile compounds were generated by the reaction of glucose and histidine and 10 volatile compounds were generated by the reaction of glucose and lysine. Reactions of ethanal with lysine and with histidine both generated 31 volatile compounds that contributed to the flavor of Jinhua ham. This indicates that histidine and lysine related to Maillard reactions possibly play important roles in the generation of the unique flavor compounds in Jinhua ham. This research demonstrates that free amino acids participate in the generation of volatile compounds from Jinhua ham via the Maillard reaction and provides a basic mechanism to explain flavor formation in Jinhua ham. Jinhua ham is a well-known traditional Chinese dry-cured meat product. However, the formation of the compounds comprising its special flavor is not well understood. Our results indicate that Maillard reactions occur in Jinhua ham under ripening conditions. This work illustrates the contribution of Maillard reactions to the flavor of Jinhua ham. © 2017 Institute of Food Technologists®.

  7. Quadratic residues and non-residues selected topics

    CERN Document Server

    Wright, Steve

    2016-01-01

    This book offers an account of the classical theory of quadratic residues and non-residues with the goal of using that theory as a lens through which to view the development of some of the fundamental methods employed in modern elementary, algebraic, and analytic number theory. The first three chapters present some basic facts and the history of quadratic residues and non-residues and discuss various proofs of the Law of Quadratic Reciprosity in depth, with an emphasis on the six proofs that Gauss published. The remaining seven chapters explore some interesting applications of the Law of Quadratic Reciprocity, prove some results concerning the distribution and arithmetic structure of quadratic residues and non-residues, provide a detailed proof of Dirichlet’s Class-Number Formula, and discuss the question of whether quadratic residues are randomly distributed. The text is a valuable resource for graduate and advanced undergraduate students as well as for mathematicians interested in number theory.

  8. Nanomechanics of the Cartilage Extracellular Matrix

    Science.gov (United States)

    Han, Lin; Grodzinsky, Alan J.; Ortiz, Christine

    2011-08-01

    Cartilage is a hydrated biomacromolecular fiber composite located at the ends of long bones that enables proper joint lubrication, articulation, loading, and energy dissipation. Degradation of extracellular matrix molecular components and changes in their nanoscale structure greatly influence the macroscale behavior of the tissue and result in dysfunction with age, injury, and diseases such as osteoarthritis. Here, the application of the field of nanomechanics to cartilage is reviewed. Nanomechanics involves the measurement and prediction of nanoscale forces and displacements, intra- and intermolecular interactions, spatially varying mechanical properties, and other mechanical phenomena existing at small length scales. Experimental nanomechanics and theoretical nanomechanics have been applied to cartilage at varying levels of material complexity, e.g., nanoscale properties of intact tissue, the matrix associated with single cells, biomimetic molecular assemblies, and individual extracellular matrix biomolecules (such as aggrecan, collagen, and hyaluronan). These studies have contributed to establishing a fundamental mechanism-based understanding of native and engineered cartilage tissue function, quality, and pathology.

  9. Extracellular signaling and multicellularity in Bacillus subtilis.

    Science.gov (United States)

    Shank, Elizabeth Anne; Kolter, Roberto

    2011-12-01

    Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Role of extracellular vesicles in rheumatoid arthritis.

    Science.gov (United States)

    Fu, Haitao; Hu, Die; Zhang, Licheng; Tang, Peifu

    2018-01-01

    Cell-derived extracellular vesicles (EVs) are involved in the pathogenesis of rheumatoid arthritis (RA), playing important roles in antigen presentation, inflammation, angiogenesis, cell-cell signal communication, thrombosis, and articular cartilage extracellular matrix degradation. Understanding the pathogenic mechanism of RA is important for developing therapies. The pathogenic indicators of RA, such as submicron-sized EVs, represent promising biomarkers for evaluating RA activity. This review summarizes the recent advances in understanding the pathogenesis of RA, and sheds light on the pathogenic as well as anti-inflammatory or immunosuppressive roles of EVs. We suggest that EVs could be harnessed as tools for drug delivery or targets for RA therapies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

    Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

  12. Apoptotic Bodies: Selective Detection in Extracellular Vesicles.

    Science.gov (United States)

    Hauser, Paul; Wang, Sha; Didenko, Vladimir V

    2017-01-01

    Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.The approach exclusively labels the vesicles that contain DNA with 5'PO 4 blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.

  13. Extracellular Matrix Biomarkers for Diagnosis, Prognosis, Imaging, and Targeting

    Science.gov (United States)

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0240 TITLE: Extracellular Matrix Biomarkers for Diagnosis, Prognosis, Imaging, and Targeting PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE Extracellular Matrix Biomarkers for Diagnosis, Prognosis, Imaging, and Targeting 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14...the management and treatment of metastatic breast cancer. 15. SUBJECT TERMS Breast Cancer, Metastasis, Extracellular Matrix , Tumor Microenvironment

  14. Molecular mechanism of Zn2+ agonism in the extracellular domain of GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    Ala substitution of potential metal-ion binding residues in the main ligand-binding pocket of the Zn2+-activated G protein-coupled receptor 39 (GPR39) receptor did not decrease Zn2+ potency. In contrast, Zn2+ stimulation was eliminated by combined substitution of His17 and His19, located in the N......-terminal segment. Surprisingly, substitution of Asp313 located in extracellular loop 3 greatly increased ligand-independent signaling and apparently eliminated Zn2+-induced activation. It is proposed that Zn2+ acts as an agonist for GPR39, not in the classical manner by directly stabilizing an active conformation...

  15. A Novel Secretory Poly-Cysteine and Histidine-Tailed Metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

    Science.gov (United States)

    Radoslavov, Georgi; Jordanova, Rositsa; Teofanova, Denitsa; Georgieva, Katya; Hristov, Petar; Salomone-Stagni, Marco; Liebau, Eva; Bankov, Ilia

    2010-01-01

    Background Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite - host interactions. Methodology/Principal Findings In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1∶2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite. Conclusions/Significance Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future. PMID:20967224

  16. A novel secretory poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP from Trichinella spiralis (Nematoda.

    Directory of Open Access Journals (Sweden)

    Georgi Radoslavov

    Full Text Available BACKGROUND: Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP. The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1:2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future.

  17. Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

    Science.gov (United States)

    Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

    2016-02-09

    β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

  18. A CHASE3/GAF sensor hybrid histidine kinase BmsA modulates biofilm formation and motility in Pseudomonas alkylphenolica.

    Science.gov (United States)

    Lee, Kyoung; Ha, Gwang Su; Veeranagouda, Yaligara; Seo, Young-Su; Hwang, Ingyu

    2016-11-01

    Pseudomonas alkylphenolica is an important strain in the biodegradation of toxic alkylphenols and mass production of bioactive polymannuronate polymers. This strain forms a diverse, 3D biofilm architecture, including mushroom-like aerial structures, circular pellicles and surface spreading, depending on culture conditions. A mutagenesis and complementation study showed that a predicted transmembrane kinase, PSAKL28_21690 (1164 aa), harbouring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic GAF, histidine kinase and three CheY-like response regulator domains, plays a positive role in the formation of the special biofilm architecture and a negative role in swimming activity. In addition, the gene, named here as bmsA, is co-transcribed with three genes encoding proteins with CheR (PSAKL28_21700) and CheB (PSAKL28_21710) domains and response regulator and histidine kinase domains (PSAKL28_21720). This gene cluster is thus named bmsABCD and is found widely distributed in pseudomonads and other bacteria. Deletion of the genes in the cluster, except forbmsA, did not result in changes in biofilm-related phenotypes. The RNA-seq analysis showed that the expression of genes coding for flagellar synthesis was increased when bmsA was mutated. In addition, the expression of rsmZ, which is one of final targets of the Gac regulon, was not significantly altered in the bmsA mutant, and overexpression of bmsA in the gacA mutant did not produce the WT phenotype. These results indicate that the sensory Bms regulon does not affect the upper cascade of the Gac signal transduction pathway for the biofilm-related phenotypes in P. alkylphenolica.

  19. Imaging Extracellular Protein Concentration with Nanoplasmonic Sensors

    OpenAIRE

    Byers, Jeff M.; Christodoulides, Joseph A.; Delehanty, James B.; Raghu, Deepa; Raphael, Marc P.

    2015-01-01

    Extracellular protein concentrations and gradients queue a wide range of cellular responses, such as cell motility and division. Spatio-temporal quantification of these concentrations as produced by cells has proven challenging. As a result, artificial gradients must be introduced to the cell culture to correlate signal and response. Here we demonstrate a label-free nanoplasmonic imaging technique that can directly map protein concentrations as secreted by single cells in real time and which ...

  20. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (inflammation in immune cells. © 2017 American Heart Association, Inc.

  1. Neutrophil extracellular traps in ischemic stroke thrombi.

    Science.gov (United States)

    Laridan, Elodie; Denorme, Frederik; Desender, Linda; François, Olivier; Andersson, Tommy; Deckmyn, Hans; Vanhoorelbeke, Karen; De Meyer, Simon F

    2017-08-01

    Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular, no information is yet available on the presence of NETs in cerebral occlusions. Such information is, however, essential to improve current thrombolytic therapy with tissue plasminogen activator (t-PA). This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi. Sixty-eight thrombi retrieved from ischemic stroke patients undergoing endovascular treatment were characterized by immunostaining using neutrophil markers (CD66b and neutrophil elastase) and NET markers (citrullinated histone H3 [H3Cit] and extracellular DNA). Neutrophils and NETs were quantified. In addition, extracellular DNA was targeted by performing ex vivo lysis of retrieved thrombi with DNase 1 and t-PA. Neutrophils were detected extensively throughout all thrombi. H3Cit, a hallmark of NETs, was observed in almost all thrombi. H3Cit-positive area varied up to 13.45% of total thrombus area. Colocalization of H3Cit with extracellular DNA released from neutrophils confirmed the specific presence of NETs. H3Cit was more abundant in thrombi of cardioembolic origin compared to other etiologies. Older thrombi contained significantly more neutrophils and H3Cit compared to fresh thrombi. Interestingly, ex vivo lysis of patient thrombi was more successful when adding DNase 1 to standard t-PA. Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. Ann Neurol 2017;82:223-232. © 2017 American Neurological Association.

  2. Circulating Extracellular RNA Markers of Liver Regeneration.

    Directory of Open Access Journals (Sweden)

    Irene K Yan

    Full Text Available Although a key determinant of hepatic recovery after injury is active liver regeneration, the ability to detect ongoing regeneration is lacking. The restoration of liver mass after hepatectomy involves systemic changes with coordinated changes in gene expression guiding regenerative responses, activation of progenitor cells, and proliferation of quiescent hepatocytes. We postulated that these responses involve intercellular communication involving extracellular RNA and that these could represent biomarkers of active regenerative responses.RNA sequencing was performed to identify temporal changes in serum extracellular non-coding RNA after partial hepatectomy in C57BL/6 male mice. Tissue expression of selected RNA was performed by microarray analysis and validated using qRT-PCR. Digital PCR was used to detect and quantify serum expression of selected RNA.A peak increase in extracellular RNA content occurred six hours after hepatectomy. RNA sequencing identified alterations in several small non-coding RNA including known and novel microRNAs, snoRNAs, tRNA, antisense and repeat elements after partial hepatectomy. Combinatorial effects and network analyses identified signal regulation, protein complex assembly, and signal transduction as the most common biological processes targeted by miRNA that altered. miR-1A and miR-181 were most significantly altered microRNA in both serum and in hepatic tissues, and their presence in serum was quantitated using digital PCR.Extracellular RNA selectively enriched during acute regeneration can be detected within serum and represent biomarkers of ongoing liver regeneration in mice. The ability to detect ongoing active regeneration would improve the assessment of hepatic recovery from liver injury.

  3. Extracellular proteolytic activity of Deinococcus geothermalis ...

    African Journals Online (AJOL)

    Production of extracellular protease by extremophilic bacteria Deinococcus geothermalis cultivated in liquid media containing 0.1% (w/v) of peptone K, 0.1% yeast extract and 0.2% marine salt reached a maximum in 14 h of the cell growth at 45°C and pH 8.0. The enzyme was purified by a two-step procedure using ...

  4. Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

    OpenAIRE

    Aizea Morales-Kastresana; Bill Telford; Musich, Thomas A.; Katherine McKinnon; Cassandra Clayborne; Zach Braig; Ari Rosner; Thorsten Demberg; Watson, Dionysios C.; Karpova, Tatiana S.; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Pavlakis, George N.; Masaki Terabe; Marjorie Robert-Guroff

    2017-01-01

    Extracellular vesicles (EVs), including exosomes and microvesicles, are 30?800?nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be brig...

  5. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

    Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

  6. Bioinformatics Tools for Extracellular Vesicles Research.

    Science.gov (United States)

    Keerthikumar, Shivakumar; Gangoda, Lahiru; Gho, Yong Song; Mathivanan, Suresh

    2017-01-01

    Extracellular vesicles (EVs) are a class of membranous vesicles that are released by multiple cell types into the extracellular environment. This unique class of extracellular organelles which play pivotal role in intercellular communication are conserved across prokaryotes and eukaryotes. Depending upon the cell origin and the functional state, the molecular cargo including proteins, lipids, and RNA within the EVs are modulated. Owing to this, EVs are considered as a subrepertoire of the host cell and are rich reservoirs of disease biomarkers. In addition, the availability of EVs in multiple bodily fluids including blood has created significant interest in biomarker and signaling research. With the advancement in high-throughput techniques, multiple EV studies have embarked on profiling the molecular cargo. To benefit the scientific community, existing free Web-based resources including ExoCarta, EVpedia, and Vesiclepedia catalog multiple datasets. These resources aid in elucidating molecular mechanism and pathophysiology underlying different disease conditions from which EVs are isolated. Here, the existing bioinformatics tools to perform integrated analysis to identify key functional components in the EV datasets are discussed.

  7. Examination of correlation between histidine and nickel absorption by Morus L., Robinia pseudoacacia L. and Populus nigra L. using HPLC-MS and ICP-MS.

    Science.gov (United States)

    Ozen, Sukran Akkus; Yaman, Mehmet

    2016-08-02

    In this study, HPLC-MS and ICP-MS methods were used for the determination of histidine and nickel in Morus L., Robinia pseudoacacia L., and Populus nigra L. leaves taken from industrial areas including Gaziantep and Bursa cities. In the determination of histidine by HPLC-MS, all of the system parameters such as flow rate of mobile phase, fragmentor potential, injection volume and column temperature were optimized and found to be 0.2 mL min(-1), 70 V, 15 µL, and 20°C, respectively. Under the optimum conditions, histidine was extracted from plant sample by distilled water at 90°C for 30 min. Concentrations of histidine as mg kg(-1) were found to be between 2-9 for Morus L., 6-13 for Robinia pseudoacacia L., and 2-10 for Populus nigra L. Concentrations of nickel were in the ranges of 5-10 mg kg(-1) for Morus L., 3-10 mg kg(-1) for Robinia pseudoacacia L., and 0.6-4 mg kg(-1) for Populus nigra L. A significant linear correlation (r = 0.78) between histidine and Ni was observed for Populus nigra L., whereas insignificant linear correlation for Robinia pseudoacacia L. (r = 0.22) were seen. Limits of detection (LOD) and quantitation (LOQ) were found to be 0.025 mg Ni L(-1) and 0.075 mg Ni L(-1), respectively.

  8. Bioenergy from sisal residues

    Energy Technology Data Exchange (ETDEWEB)

    Jungersen, G. [Dansk Teknologisk Inst. (Denmark); Kivaisi, A.; Rubindamayugi, M. [Univ. of Dar es Salaam (Tanzania, United Republic of)

    1998-05-01

    The main objectives of this report are: To analyse the bioenergy potential of the Tanzanian agro-industries, with special emphasis on the Sisal industry, the largest producer of agro-industrial residues in Tanzania; and to upgrade the human capacity and research potential of the Applied Microbiology Unit at the University of Dar es Salaam, in order to ensure a scientific and technological support for future operation and implementation of biogas facilities and anaerobic water treatment systems. The experimental work on sisal residues contains the following issues: Optimal reactor set-up and performance; Pre-treatment methods for treatment of fibre fraction in order to increase the methane yield; Evaluation of the requirement for nutrient addition; Evaluation of the potential for bioethanol production from sisal bulbs. The processing of sisal leaves into dry fibres (decortication) has traditionally been done by the wet processing method, which consumes considerable quantities of water and produces large quantities of waste water. The Tanzania Sisal Authority (TSA) is now developing a dry decortication method, which consumes less water and produces a waste product with 12-15% TS, which is feasible for treatment in CSTR systems (Continously Stirred Tank Reactors). (EG)

  9. Time resolved and label free monitoring of extracellular metabolites by surface enhanced Raman spectroscopy.

    Directory of Open Access Journals (Sweden)

    Victoria Shalabaeva

    Full Text Available Metabolomics is an emerging field of cell biology that aims at the comprehensive identification of metabolite levels in biological fluids or cells in a specific functional state. Currently, the major tools for determining metabolite concentrations are mass spectrometry coupled with chromatographic techniques and nuclear magnetic resonance, which are expensive, time consuming and destructive for the samples. Here, we report a time resolved approach to monitor metabolite dynamics in cell cultures, based on Surface Enhanced Raman Scattering (SERS. This method is label-free, easy to use and provides the opportunity to simultaneously study a broad range of molecules, without the need to process the biological samples. As proof of concept, NIH/3T3 cells were cultured in vitro, and the extracellular medium was collected at different time points to be analyzed with our engineered SERS substrates. By identifying individual peaks of the Raman spectra, we showed the simultaneous detection of several components of the conditioned medium, such as L-tyrosine, L-tryptophan, glycine, L-phenylalanine, L-histidine and fetal bovine serum proteins, as well as their intensity changes during time. Furthermore, analyzing the whole Raman data set with the Principal Component Analysis (PCA, we demonstrated that the Raman spectra collected at different days of culture and clustered by similarity, described a well-defined trajectory in the principal component plot. This approach was then utilized to determine indirectly the functional state of the macrophage cell line Raw 264.7, stimulated with the lipopolysaccharide (LPS for 24 hours. The collected spectra at different time points, clustered by the PCA analysis, followed a well-defined trajectory, corresponding to the functional change of cells toward the activated pro-inflammatory state induced by the LPS. This study suggests that our engineered SERS surfaces can be used as a versatile tool both for the characterization

  10. Extracellular polymeric substances are transient media for microbial extracellular electron transfer

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, Enhua; Zhang, Jingdong

    2017-01-01

    Microorganisms exploit extracellular electron transfer (EET) in growth and information exchange with external environments or with other cells. Every microbial cell is surrounded by extracellular polymeric substances (EPS). Understanding the roles of three-dimensional (3D) EPS in EET is essential...... in microbiology and microbial exploitation for mineral bio-respiration, pollutant conversion, and bioenergy production. We have addressed these challenges by comparing pure and EPS-depleted samples of three representative electrochemically active strains viz Gram-negative Shewanella oneidensis MR-1, Gram...

  11. Regulation of Corneal Stroma Extracellular Matrix Assembly

    Science.gov (United States)

    Chen, Shoujun; Mienaltowski, Michael J.; Birk, David E.

    2014-01-01

    The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. PMID:25819456

  12. Nano-TiO2 affects Cu speciation, extracellular enzyme activity, and bacterial communities in sediments.

    Science.gov (United States)

    Fan, Wenhong; Liu, Tong; Li, Xiaomin; Peng, Ruishuang; Zhang, Yilin

    2016-11-01

    In aquatic ecosystems, titanium dioxide nanoparticles (nano-TiO2) coexist with heavy metals and influence the existing forms and toxicities of the metal in water. However, limited information is available regarding the ecological risk of this coexistence in sediments. In this study, the effect of nano-TiO2 on Cu speciation in sediments was investigated using sequential extraction. The microcosm approach was also employed to analyze the effects of the coexistence of nano-TiO2 and Cu on extracellular enzyme activity and bacterial communities in sediments. Results showed that nano-TiO2 decreased the organic matter-bound fraction of Cu and increased the corresponding residual fraction Cu. As a result, speciation of exogenous Cu in sediments changed. During the course of the 30-day experiment, the presence of nano-TiO2 did not affect Cu-induced changes in bacterial community structure. However, the coexistence of nano-TiO2 and Cu restrained the activity of bacterial extracellular enzymes, such as alkaline phosphatase and β-glucosidase. The degree of inhibition also varied because of the different properties of extracellular enzymes. This research highlighted the importance of understanding and predicting the effects of the coexistence of nanomaterials and other pollutants in sediments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Managing Brain Extracellular K(+) during Neuronal Activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    isoform compositions of the Na(+)/K(+)-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K(+) from neurons, whereas the neurons themselves become the primary K...... characteristics required to fulfill their distinct physiological roles in clearance of K(+) from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na(+)/K(+)-ATPase isoform combinations in K(+) management in the central nervous system might...

  14. Role of extracellular vesicles in autoimmune diseases.

    Science.gov (United States)

    Turpin, Delphine; Truchetet, Marie-Elise; Faustin, Benjamin; Augusto, Jean-François; Contin-Bordes, Cécile; Brisson, Alain; Blanco, Patrick; Duffau, Pierre

    2016-02-01

    Extracellular vesicles (EVs) consist of exosomes released upon fusion of multivesicular bodies with the cell plasma membrane and microparticles shed directly from the cell membrane of many cell types. EVs can mediate cell-cell communication and are involved in many processes including inflammation, immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. Accumulating evidence reveals that EVs act in the establishment, maintenance and modulation of autoimmune processes among several others involved in cancer and cardiovascular complications. EVs could also present biomedical applications, as disease biomarkers and therapeutic targets or agents for drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Material Utilization of Organic Residues.

    Science.gov (United States)

    Peinemann, Jan Christoph; Pleissner, Daniel

    2018-02-01

    Each year, 1.3 billion tons of food waste is generated globally. This waste traces back to industrial and agricultural producers, bakeries, restaurants, and households. Furthermore, lignocellulosic materials, including grass clippings, leaves, bushes, shrubs, and woods, appear in large amounts. Depending on the region, organic waste is either composted, burned directly, or converted into biogas. All of the options set aside the fact that organic residues are valuable resources containing carbohydrates, lipids, proteins, and phosphorus. Firstly, it is clear that avoidance of organic residues is imperative. However, the residues that accumulate nonetheless should be utilized by material means before energy production is targeted. This review presents different processes for the microbial utilization of organic residues towards compounds that are of great importance for the bioeconomy. The focus thereby is on the challenges coming along with downstream processing when the utilization of organic residues is carried out decentralized. Furthermore, a future process for producing lactic acid from organic residues is sketched.

  16. NMR spectroscopic characterization of the sialyltransferase CstII from Campylobacter jejuni: histidine 188 is the general base.

    Science.gov (United States)

    Chan, Patrick H W; Lairson, Luke L; Lee, Ho Jun; Wakarchuk, Warren W; Strynadka, Natalie C J; Withers, Stephen G; McIntosh, Lawrence P

    2009-12-01

    Cell surface glycans are often terminated by sialic acid, which is incorporated onto sugar acceptors by sialyltransferases. The crystal structure of the GT family 42 Campylobacter jejuni alpha-2,3/2,8-sialyltransferase (CstII) provides key insights into the sialyl-transfer mechanism, including tentative identification of His188 as the catalytic base. In support of this hypothesis, the CstII-H188A mutant is able to catalyze sialyl transfer from CMP-Neu5Ac to added anions such as azide and formate but not to its natural sugar acceptor lactose. Complementing this work, NMR spectroscopy was used to investigate the structure and dynamics of CstII and to measure the intrinsic pK(a) value of His188 for comparison with the pK(a) determined from the pH-dependent k(cat)/K(M) of the enzyme. By systematically introducing point mutations at the subunit interfaces, two active monomeric variants, CstII-F121D and CstII-Y125Q, were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality (1)H/(15)N-HSQC and (1)H/(13)C-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the (1)H/(15)N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a millisecond to microsecond time scale. The histidine pK(a) values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of [(13)C(epsilon1)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value approximately 6.6 of His188 matches the apparent pK(a) value approximately 6.5 governing the pH dependence of k(cat)/K(M) for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.

  17. Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes.

    Science.gov (United States)

    Larsen, R W; Nunez, D J; Morgan, W T; Muhoberac, B B; Ondrias, M R

    1992-04-01

    Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and

  18. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    Directory of Open Access Journals (Sweden)

    Chad J Johnson

    2016-09-01

    Full Text Available Neutrophils release extracellular traps (NETs in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA and was associated with suppression of neutrophil reactive oxygen species (ROS production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  19. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

  20. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

  1. A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.

    Science.gov (United States)

    Pérez-González, Rocío; Gauthier, Sebastien A; Kumar, Asok; Saito, Mitsuo; Saito, Mariko; Levy, Efrat

    2017-01-01

    Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.

  2. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Cecilia Lässer

    2016-12-01

    Full Text Available The International Society for Extracellular Vesicles (ISEV has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs. This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

  3. Cardiac Physiology of Aging: Extracellular Considerations.

    Science.gov (United States)

    Horn, Margaux A

    2015-07-01

    Aging is a major risk factor for the development of cardiovascular disease, with the majority of affected patients being elderly. Progressive changes to myocardial structure and function occur with aging, often in concert with underlying pathologies. However, whether chronological aging results in a remodeled "aged substrate" has yet to be established. In addition to myocyte contractility, myocardial performance relies heavily on the cardiac extracellular matrix (ECM), the roles of which are as dynamic as they are significant; including providing structural integrity, assisting in force transmission throughout the cardiac cycle and acting as a signaling medium for communication between cells and the extracellular environment. In the healthy heart, ECM homeostasis must be maintained, and matrix deposition is in balance with degradation. Consequently, alterations to, or misregulation of the cardiac ECM has been shown to occur in both aging and in pathological remodeling with disease. Mounting evidence suggests that age-induced matrix remodeling may occur at the level of ECM control; including collagen synthesis, deposition, maturation, and degradation. Furthermore, experimental studies using aged animal models not only suggest that the aged heart may respond differently to insult than the young, but the identification of key players specific to remodeling with age may hold future therapeutic potential for the treatment of cardiac dysfunction in the elderly. This review will focus on the role of the cardiac interstitium in the physiology of the aging myocardium, with particular emphasis on the implications to age-related remodeling in disease. © 2015 American Physiological Society.

  4. [Glutamic acid as a universal extracellular signal].

    Science.gov (United States)

    Yoneda, Yukio

    2015-08-01

    The prevailing view is that both glutamic (Glu) and gamma-aminobutyric (GABA) acids play a role as an amino acid neurotransmitter released from neurons. However, little attention has been paid to the possible expression and functionality of signaling machineries required for amino acidergic neurotransmission in cells other than central neurons. In line with our first demonstration of the presence of Glu receptors outside the brain, in this review I will outline our recent findings accumulated since then on the physiological and pathological significance of neuronal amino acids as an extracellular signal essential for homeostasis in a variety of phenotypic cells. In undifferentiated neural progenitor cells, for instance, functional expression is seen with different signaling machineries used for glutamatergic and GABAergic neurotransmission in neurons. Moreover, Glu plays a role in mechanisms underlying suppression of proliferation for self-replication in undifferentiated mesenchymal stem cells. There is more accumulating evidence for neuronal amino acids playing a role as an extracellular autocrine or paracrine signal commonly used in different phenotypic cells. Evaluation of drugs currently used could be thus beneficial for the efficient prophylaxis and/or the therapy of a variety of diseases relevant to disturbance of amino acid signaling in diverse organs.

  5. Metabolism of extracellular phospholipids in Tetrahymena pyriformis.

    Science.gov (United States)

    Arai, H; Nishikawa, K; Inoue, K; Nozawa, Y; Nojima, S

    1987-05-01

    We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."

  6. Defining the extracellular matrix using proteomics

    Science.gov (United States)

    Byron, Adam; Humphries, Jonathan D; Humphries, Martin J

    2013-01-01

    The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology. PMID:23419153

  7. Brain Extracellular Space as a Diffusion Barrier.

    Science.gov (United States)

    Nicholson, Charles; Kamali-Zare, Padideh; Tao, Lian

    2011-10-01

    The extracellular space (ECS) consists of the narrow channels between brain cells together with their geometrical configuration and contents. Despite being only 20-60 nm in width, the ECS typically occupies 20% of the brain volume. Numerous experiments over the last 50 years have established that molecules moving through the ECS obey the laws of diffusion but with an effective diffusion coefficient reduced by a factor of about 2.6 compared to free diffusion. This review considers the origins of the diffusion barrier arising from the ECS and its properties. The paper presents a brief overview of software for implementing two point-source paradigms for measurements of localized diffusion properties: the real-time iontophoresis or pressure method for small ions and the integrative optical imaging method for macromolecules. Selected results are presented. This is followed by a discussion of the application of the MCell Monte Carlo simulation program to determining the importance of geometrical constraints, especially dead-space microdomains, and the possible role of interaction with the extracellular matrix. It is concluded that we can predict the impediment to diffusion of many molecules of practical importance and also use studies of the diffusion of selected molecular probes to reveal the barrier properties of the ECS.

  8. Evaluation of residue-residue contact predictions in CASP9

    KAUST Repository

    Monastyrskyy, Bohdan

    2011-01-01

    This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures being the precision in recognizing contacts and the difference between the distribution of distances in the subset of predicted contact pairs versus all pairs of residues in the structure. The emphasis is placed on the prediction of long-range contacts (i.e., contacts between residues separated by at least 24 residues along sequence) in target proteins that cannot be easily modeled by homology. Although there is considerable activity in the field, the current analysis reports no discernable progress since CASP8.

  9. Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

    Science.gov (United States)

    Luo, Ling; Meng, Han; Gu, Ji-Dong

    2017-07-15

    Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. The role of extracellular vesicles in malaria biology and pathogenesis.

    Science.gov (United States)

    Sampaio, Natalia Guimaraes; Cheng, Lesley; Eriksson, Emily M

    2017-06-09

    In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.

  11. Extracellular RNAs: development as biomarkers of human disease

    Directory of Open Access Journals (Sweden)

    Joseph F. Quinn

    2015-08-01

    Full Text Available Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined.

  12. Requirement of extracellular Ca2+binding to specific amino acids for heat-evoked activation of TRPA1.

    Science.gov (United States)

    Kurganov, Erkin; Saito, Shigeru; Tanaka Saito, Claire; Tominaga, Makoto

    2017-04-15

    We found that extracellular Ca 2+ , but not other divalent cations (Mg 2+ and Ba 2+ ) or intracellular Ca 2+ , is involved in heat-evoked activation of green anole (ga) TRPA1. Heat-evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca 2+ . Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca 2+ is important for heat-evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca 2+ , but not intracellular Ca 2+ , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca 2+ -dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat-activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca 2+ , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked TRPA1 activation in the presence of extracellular Ca 2+ . These results suggest that neutralization of acidic amino acids by extracellular Ca 2+ is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  13. Transcriptomic profiling of platelet senescence and platelet extracellular vesicles.

    Science.gov (United States)

    Pienimaeki-Roemer, Annika; Konovalova, Tatiana; Musri, Melina M; Sigruener, Alexander; Boettcher, Alfred; Meister, Gunter; Schmitz, Gerd

    2017-01-01

    Platelets (PLTs) are derived from megakaryocytes during PLT shedding. Senescent or activated PLTs are expanded in vascular and neurological diseases and release PLT extracellular vesicles (PL-EVs). A systematic analysis of regular messenger RNA (mRNA) and small RNA composition in PLTs and PL-EVs during in vitro PLT senescence has not yet been published. We isolated PLTs, total PL-EVs, and PL-EV subsets on Days 0 and 5 from human stored donor platelet concentrates. Isolated mRNA species and microRNA (miRNA) species were analyzed by microarrays and deep sequencing. Correlation of mRNA and miRNA species (miR) and miRNA target analyses were performed using bioinformatics. During in vitro PLT senescence, residual PLT mRNA species were decreased and partially converted to miRNA species. Residual mRNAs included encoded genes relevant for atherosclerosis, inflammation (matrix metallopeptidase 14 [MMP-14], granulin [GRN], angiopoietin like 2 [ANGPTL2]), and neurotransmission (dopamine receptor 2 [DRD2], γ-aminobutyric acid type A receptor ρ3 [GABRR3]). Compared with senescent PLTs, PL-EVs have up-regulated their miRNA species involved in "diabesity" and in vascular and metabolic disease (miR-144-3p, miR-486-5p, miR-142-5p, miR-451a, miR-25-3p, miR-145-5p, and let-7f-5p). The 100 highest expressed PL-EV miRNA species determined by microarrays were compared with the 100 highest expressed PL-EV miRNA species detected by deep sequencing. This approach resulted in 66 overlaps. The regulated miRNAs (assessed by both methods) were related to neurological disorders, including targets for Alzheimer's disease (e.g., β-site amyloid precursor protein APP-cleaving enzyme 1 [BACE1], translocase of outer mitochondrial membrane 40 homolog [TOMM40], neuron navigator 3 [NAV3]). During in vitro senescence, PLTs degrade large RNA species. Concomitantly, they up-regulate a distinct set of known small RNA species involved in atherosclerosis, inflammation, and neurodegeneration. PL-EVs enrich

  14. Landfilling of waste incineration residues

    DEFF Research Database (Denmark)

    Christensen, Thomas Højlund; Astrup, Thomas; Cai, Zuansi

    2002-01-01

    Residues from waste incineration are bottom ashes and air-pollution-control (APC) residues including fly ashes. The leaching of heavy metals and salts from the ashes is substantial and a wide spectrum of leaching tests and corresponding criteria have been introduced to regulate the landfilling...

  15. Residual number processing in dyscalculia

    OpenAIRE

    CAPPELLETTI, MARINELLA; Price, Cathy J.

    2014-01-01

    Developmental dyscalculia ? a congenital learning disability in understanding numerical concepts ? is typically associated with parietal lobe abnormality. However, people with dyscalculia often retain some residual numerical abilities, reported in studies that otherwise focused on abnormalities in the dyscalculic brain. Here we took a different perspective by focusing on brain regions that support residual number processing in dyscalculia. All participants accurately performed semantic and ca...

  16. Gasification of shredder residue (AUTOGAS)

    Energy Technology Data Exchange (ETDEWEB)

    Nieminen, M.; Suomalainen, M.; Maekinen, T. [VTT Processes, Espoo (Finland); Jalkanen, H.; Huitu, K. [Helsinki University of Technology, Espoo (Finland)

    2004-07-01

    The disposal of shredder residue is presently based worldwide on landfilling. However, the current trend in the legislation of European Union is to emphasise, at a first priority, material recycling and, in addition, to prefer energy recovery to landfilling. Gasification is a potential option to utilise shredder residue in energy production in environmentally acceptable way. In addition some metals in shredder residue may be recovered from gasification ash leading to increase in material recycling. An air blown fluidised bed gasification process for thermal treatment of shredder residue will be developed in the project. The process will produce fuel gas, which will be clean enough to be used as replacement of other fuels in coal fired boilers, industrial kilns or other applications. Attention is also paid on the economic feasibility of the process concept. Three authentic shredder residue samples delivered by shredders were characterised by chemical analysis and then used as a feedstock in bench- scale and PDU-scale fluidised bed gasification test trials. The results indicated that the process concept is a potential application for treating shredder residue. Based on the gasification test results, a preliminary evaluation of technological and economic feasibility of the process concept was carried out. The overall conclusion after the preliminary evaluation was that air-blown gasification of shredder residue is a very interesting and economically competitive alternative for treating shredder residue. However, there are still needs for further development of process details. (orig.)

  17. Use of Residues in Agriculture

    Directory of Open Access Journals (Sweden)

    Fabio Olivieri de Nobile

    2015-12-01

    Full Text Available This article presents a revision of the abstracts of The XX Brazilian Meeting of Fertility of the Soil and Nutrition of Plants, realizado in Piracicaba-São Paulo. The agronomic use of residues pressuposes a diverse number of applications such as their use in: animal feeding, substrate for fermentations, organic or organo-mineral fertilizer manufacture, soil coverages in different creations and substitutes of raw material for the agroindustrial or similar activities. However, the objective of this presentation is restricted to the aspects of the use of residues in the soil, understanding the aspects related to the characterization of these residues, the benefits or inconveniences of the application to the soil and the parameters that must be observed when the intention is to give this destination to the residues. In this way, this subject can be divided in two basic aspects: the residues and the soils. Concerning the residues, the main factors that affect their application to the soil are: chemical composition, physical characteristics, sanitary aspects, generated amount and regimen of release. In relation to soil, we must consider all those characteristics which are responsible for the capacity of the soil in deactivating and stabilizing the residues, through physical chemical and biological mechanisms. It is in this general framework that we intend to approach some specific aspects which are considered more important for the use of residues in soil.

  18. Comparative survival analysis of 12 histidine kinase mutants of Deinococcus radiodurans after exposure to DNA-damaging agents.

    Science.gov (United States)

    Im, Seonghun; Song, Dusup; Joe, Minho; Kim, Dongho; Park, Don-Hee; Lim, Sangyong

    2013-06-01

    Bacteria are able to adapt to changes in the environment using two-component signal transduction systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). Deinococcus radiodurans, one of the most resistant organisms to ionizing radiation, has 20 putative HKs and 25 putative RRs. In this study, we constructed 12 D. radiodurans mutant strains lacking a gene encoding a HK and surveyed their resistance to γ-radiation, UV-B radiation (302 nm), mitomycin C (MMC), and H(2)O(2). Five (dr0860 (-), dr1174 (-), dr1556 (-), dr2244 (-), and dr2419 (-)) of the 12 mutant strains showed at least a one-log cycle reduction in γ-radiation resistance. The mutations (1) dr1174, dr1227, and dr2244 and (2) dr0860, dr2416, and dr2419 caused decreases in resistance to UV radiation and MMC, respectively. Only the dr2416 and dr2419 mutant strains showed higher sensitivity to H(2)O(2) than the wild-type. Reductions in the resistance to γ-radiation and H(2)O(2), but not to UV and MMC, were observed in the absence of DR2415, which seems to be a cognate RR of DR2416. This result suggests that DR2415/DR2416 (DrtR/S: DNA damage response TCS) may be another TCS responsible for the extreme resistance of D. radiodurans to DNA-damaging agents.

  19. A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum.

    Science.gov (United States)

    Duan, Yabing; Ge, Changyan; Liu, Shengming; Wang, Jianxin; Zhou, Mingguo

    2013-09-01

    Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  20. Phosphorylation of Spo0A by the Histidine Kinase KinD Requires the Lipoprotein Med in Bacillus subtilis ▿

    Science.gov (United States)

    Banse, Allison V.; Hobbs, Errett C.; Losick, Richard

    2011-01-01

    The response regulatory protein Spo0A of Bacillus subtilis is activated by phosphorylation by multiple histidine kinases via a multicomponent phosphorelay. Here we present evidence that the activity of one of the kinases, KinD, depends on the lipoprotein Med, a mutant of which has been known to cause a cannibalism phenotype. We show that the absence of Med impaired and the overproduction of Med stimulated the transcription of two operons (sdp and skf) involved in cannibalism whose transcription is known to depend on Spo0A in its phosphorylated state (Spo0A∼P). Further, these effects of Med were dependent on KinD but not on kinases KinA, KinB, and KinC. Additionally, we show that deletion or overproduction of Med impaired or enhanced, respectively, biofilm formation and that these effects, too, depended specifically on KinD. Finally, we report that overproduction of Med bypassed the dominant negative effect on transcription of sdp of a truncated KinD retaining the transmembrane segments but lacking the kinase domain. We propose that Med directly or indirectly interacts with KinD in the cytoplasmic membrane and that this interaction is required for KinD-dependent phosphorylation of Spo0A. PMID:21622736

  1. Phosphorylation of Spo0A by the histidine kinase KinD requires the lipoprotein med in Bacillus subtilis.

    Science.gov (United States)

    Banse, Allison V; Hobbs, Errett C; Losick, Richard

    2011-08-01

    The response regulatory protein Spo0A of Bacillus subtilis is activated by phosphorylation by multiple histidine kinases via a multicomponent phosphorelay. Here we present evidence that the activity of one of the kinases, KinD, depends on the lipoprotein Med, a mutant of which has been known to cause a cannibalism phenotype. We show that the absence of Med impaired and the overproduction of Med stimulated the transcription of two operons (sdp and skf) involved in cannibalism whose transcription is known to depend on Spo0A in its phosphorylated state (Spo0A∼P). Further, these effects of Med were dependent on KinD but not on kinases KinA, KinB, and KinC. Additionally, we show that deletion or overproduction of Med impaired or enhanced, respectively, biofilm formation and that these effects, too, depended specifically on KinD. Finally, we report that overproduction of Med bypassed the dominant negative effect on transcription of sdp of a truncated KinD retaining the transmembrane segments but lacking the kinase domain. We propose that Med directly or indirectly interacts with KinD in the cytoplasmic membrane and that this interaction is required for KinD-dependent phosphorylation of Spo0A.

  2. Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

    Directory of Open Access Journals (Sweden)

    Kristin M. Priebatsch

    2017-03-01

    Full Text Available Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG, which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+. HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

  3. LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus.

    Directory of Open Access Journals (Sweden)

    Gabriela Sycz

    Full Text Available Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.

  4. Bromobenzene 3,4-oxide alkylates histidine and lysine side chains of rat liver proteins in vivo.

    Science.gov (United States)

    Bambal, R B; Hanzlik, R P

    1995-01-01

    The hepatotoxic effects of bromobenzene (BB) are correlated with and generally ascribed to the covalent modification of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. Previous studies revealed that quinone as well as epoxide metabolites of BB form adducts to protein sulfur nucleophiles, that the quinone-derived adducts are more abundant by a factor of ca. 7, and that collectively these sulfur adducts account for only about 10% of the total protein covalent binding [Slaughter, D. E., and Hanzlik, R. P. (1991) Chem. Res. Toxicol. 4, 349-359]. To examine the possibility that metabolically-formed BB-3,4-oxide alkylates nitrogen nucleophiles on proteins under toxicologically relevant conditions in vivo, we synthesized standards of N tau-(p-bromophenyl)histidine (7) and N epsilon-(p-bromophenyl)lysine (8) as anticipated adduct structures and used them to guide a chromatographic search for their presence in hydrolysates of liver protein from BB-treated rats. While radio-LC chromatography and GC/MS provide unequivocal evidence for their presence, the amounts of 7 and 8 observed are very low ( covalent binding). The apparently small net contribution of epoxide metabolites to covalent binding of BB in vivo suggests the majority of binding may arise via quinone metabolites, but this should not be construed to imply that quinone adducts are necessarily more important toxicologically than epoxide adducts; in this context the identity of the protein targets is probably at least as important as the type of electrophilic metabolite involved.

  5. Effect of Ni2+ Doping on the Growth and Properties of Mn-L-Histidine Hydrochloride Monohydrate Crystals

    Directory of Open Access Journals (Sweden)

    J. Sai Chandra

    2013-01-01

    Full Text Available The main focus of this work had been to grow good quality crystals from amino acids and amino acid-based materials for nonlinear optics (NLO applications. For the first time, a series of amino acid complexes doped with transition metal ions were grown in our laboratory from aqueous solutions by slow evaporation technique. Ni(II ion doped Manganese L-Histidine hydrochloride monohydrate (Ni(II-MnLHICl crystals were grown on the same lines and were characterized by powder X-ray diffraction (XRD, optical absorption, electron paramagnetic resonance, and infrared absorption studies. From Powder XRD, the unit cell lattice parameters were calculated as a=1.5301 nm, b=0.8928 nm and c=0.6851 nm. From electron paramagnetic resonance (EPR spectra, isotropic “g” factor and spin hamiltonian parameter A all were calculated as 2.0439 and 20×10−4, respectively. From optical absorption studies, crystal field splitting value (Dq and the interelectron repulsion parameters B and C were calculated for Ni2+ and Mn2+ as Dq=850 cm−1, B=725 cm−1, C=2640 cm−1 and Dq=915 cm−1, B=810 cm−1, C=2780 cm−1, respectively. The presence of various functional groups and the modes of vibrations were confirmed by FTIR studies.

  6. Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

    Science.gov (United States)

    Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

    2013-01-01

    The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

  7. [Interaction between folate deficiency and aberrant expression related to fragile histidine triad gene in the progression of cervical cancerization].

    Science.gov (United States)

    Chen, Xiao; Wang, Jintao; Bai, Lixia; Ding, Ling; Wu, Tingting; Bai, Lan; Xu, Juan; Sun, Xuesong

    2015-04-01

    To explore the interaction between folate deficiency and aberrant expression related to fragile histidine triad (FHIT) gene in the progression of cervical cancerization. A total number of 80 patients with histological diagnosis of cervix inflammation (CI), 55 cervical intraepithelial neoplasm I (CIN I), 55 cervical intraepithelial neoplasm II/III (CIN II/III) and 64 cervical squamous cell carcinoma (SCC) were included in this study. Levels of serum folate were detected by microbiological assay method and the methylation status of FHIT gene CpG islands was tested by methylation-specific PCR (MSP). FHIT protein levels were measured by Western blot. In vitro, cervical cancer cell lines CaSki (HPV16-positive) was treated with different concentrations of folate. Proliferation and apoptosis of cells, methylation of FHIT gene and the levels of FHIT protein expression were measured in each group. All analyses were performed with SPSS (version 17.0) statistical software. Differences among groups were assessed by chi-square test, Kruskal-Wallis test. Spearman correlation, and the interaction effects were evaluated by additive model. The levels of serum folate (H = 59.08, P cancer and cervix precancerous lesions, and thus play a synergistic action in the progression of cervical cancerization.

  8. Novel sigmaB regulation modules of Gram-positive bacteria involve the use of complex hybrid histidine kinases.

    Science.gov (United States)

    de Been, Mark; Francke, Christof; Siezen, Roland J; Abee, Tjakko

    2011-01-01

    A common bacterial strategy to cope with stressful conditions is the activation of alternative sigma factors that control specific regulons enabling targeted responses. In the human pathogen Bacillus cereus, activation of the major stress-responsive sigma factor σ(B) is controlled by a signalling route that involves the multi-sensor hybrid histidine kinase RsbK. RsbK-type kinases are not restricted to the B. cereus group, but occur in a wide variety of other bacterial species, including members of the the low-GC Gram-positive genera Geobacillus and Paenibacillus as well as the high-GC actinobacteria. Genome context and protein sequence analyses of 118 RsbK homologues revealed extreme variability in N-terminal sensory as well as C-terminal regulatory domains and suggested that RsbK-type kinases are subject to complex fine-tuning systems, including sensitization and desensitization via methylation and demethylation within the helical domain preceding the H-box. The RsbK-mediated stress-responsive sigma factor activation mechanism that has evolved in B. cereus and the other species differs markedly from the extensively studied and highly conserved RsbRST-mediated σ(B) activation route found in Bacillus subtilis and other low-GC Gram-positive bacteria. Implications for future research on sigma factor control mechanisms are presented and current knowledge gaps are briefly discussed.

  9. Screening for Methylated Poly(⌊-histidine with Various Dimethylimidazolium/Methylimidazole/Imidazole Contents as DNA Carrier

    Directory of Open Access Journals (Sweden)

    Shoichiro Asayama

    2015-08-01

    Full Text Available Methylated poly(l-histidine (PLH-Me, our original polypeptide, has controlled the contents of dimethylimidazolium, τ/π-methylimidazole and imidazole groups for efficient gene delivery. The screening for the PLH-Me as DNA carrier has been carried out by use of the PLH with 25 mol% (τ-methyl, 16 mol%; π-methyl, 17 mol%; deprotonated imidazole, 41 mol%, 68 mol% (τ-methyl, 16 mol%; π-methyl, 8 mol%; deprotonated imidazole, 8 mol% and 87 mol% (τ-methyl, 7 mol%; π-methyl, 4 mol%; deprotonated imidazole, 2 mol% dimethylimidazolium groups, that is, PLH-Me(25, PLH-Me(68 and PLH-Me(87, respectively. The screening of the chemical structure of PLH-Me has been carried out for DNA carrier properties, which are the stability of its DNA polyion complexes and gene expression. The DNA complexes with the 25 mol% and 68 mol% dimethylated PLH-Me possessed almost same ability to retain DNA, as compared with the 87 mol% dimethylated PLH-Me, which was examined by competitive exchange with dextran sulfate. From the gene transfection experiment against HepG2 cells, human hepatoma cell line, the PLH-Me(25/DNA complex was revealed to mediate highest gene expression. These results suggest that the dimethyl-imidazolium/methylimidazole/imidazole balance of the PLH-Me is important for DNA carrier design.

  10. The histidine-phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system of Bacillus sphaericus self-associates.

    Directory of Open Access Journals (Sweden)

    Rosa Doménech

    Full Text Available The phosphotransferase system (PTS is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI and the histidine phosphocarrier (HPr. In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPr(bs, and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPr(bs forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EIN(sc, with a higher affinity than that of the natural partner of EIN(sc, HPr(sc. Modelling of the complex between EIN(sc and HPr(bs suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPr(bs for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPr(sc and EIN(sc.

  11. The histidine-phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system of Bacillus sphaericus self-associates.

    Science.gov (United States)

    Doménech, Rosa; Hernández-Cifre, José G; Bacarizo, Julio; Díez-Peña, Ana I; Martínez-Rodríguez, Sergio; Cavasotto, Claudio N; de la Torre, José García; Cámara-Artigás, Ana; Velázquez-Campoy, Adrián; Neira, José L

    2013-01-01

    The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI) and the histidine phosphocarrier (HPr). In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPr(bs), and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPr(bs) forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EIN(sc), with a higher affinity than that of the natural partner of EIN(sc), HPr(sc). Modelling of the complex between EIN(sc) and HPr(bs) suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPr(bs) for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPr(sc) and EIN(sc).

  12. Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents

    OpenAIRE

    Verma, Priyanka; Biswas, Sukla; Mohan, Teena; Ali, Shakir; Rao, D.N.

    2013-01-01

    Background & objectives: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antige...

  13. NikA/TcsC histidine kinase is involved in conidiation, hyphal morphology, and responses to osmotic stress and antifungal chemicals in Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Daisuke Hagiwara

    Full Text Available The fungal high osmolarity glycerol (HOG pathway is composed of a two-component system (TCS and Hog1-type mitogen-activated protein kinase (MAPK cascade. A group III (Nik1-type histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

  14. Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy

    NARCIS (Netherlands)

    Nuland, Nico A.J. van; Grötzinger, Joachim; Dijkstra, Klaas; Scheek, Ruud M.; Robillard, George T.

    1992-01-01

    We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments to the side-chain 1H, 15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE

  15. Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803

    OpenAIRE

    Kuwahara, Ayuko; Arisaka, Satomi; Takeya, Masahiro; Iijima, Hiroko; Hirai, Masami Yokota; Osanai, Takashi

    2015-01-01

    Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the ...

  16. EFFECT OF Β-ALANINE AND L-HISTIDINE ON CONCENTRATION OF CARNOSINE IN MUSCLE TISSUE AND OXIDATIVE STABILITY OF CHICKEN MEAT

    Directory of Open Access Journals (Sweden)

    Gordana Kralik

    2015-09-01

    Full Text Available This paper presents the results of two separate experiments, each involving 75 chickens of Cobb 500 provenience, divided into three experimental groups. During the last three weeks of fattening, chickens were fed finisher diets supplemented with amino acids β-alanine (0%, 0.5% and 1% and L-histidine (0%, 0.3% and 0.5% in different portions. After chickens have been slaughtered, 10 samples of breast tissue were taken from each group for carnosine content determination in muscle tissue and lipid oxidation expressed as TBARS. Analysis of THE results referring to carnosine concentration in breast muscle proved that supplementation of 0.5% L-histidine affected the carnosine concentration increase in breast muscles from 941.58 µg/g of tissue (H1 to 1186.06 µg/g of tissue (H3, while supplementation of 1% β-alanine influenced the increase in carnosine concentration from 756.15 µg/g of tissue (A1 to 911.01 µg/g of tissue (A3. Supplementation of amino acids did not have effects on TBARS values, but oxidation values decreased along with the supplementation of higher amounts of amino acids to diets, which was particularly expressed in samples stored for 60 days at -20°C. The experimental group H3 (0.5% L-histidine exhibited 30.54% lower value of lipid oxidation than the control one H1 (0% L-histidine, while the group with 1% β-alanine (A3 had lipid oxidation value by 17.65% lower than the control group A1 (0% β-alanine.

  17. Statistical inference on residual life

    CERN Document Server

    Jeong, Jong-Hyeon

    2014-01-01

    This is a monograph on the concept of residual life, which is an alternative summary measure of time-to-event data, or survival data. The mean residual life has been used for many years under the name of life expectancy, so it is a natural concept for summarizing survival or reliability data. It is also more interpretable than the popular hazard function, especially for communications between patients and physicians regarding the efficacy of a new drug in the medical field. This book reviews existing statistical methods to infer the residual life distribution. The review and comparison includes existing inference methods for mean and median, or quantile, residual life analysis through medical data examples. The concept of the residual life is also extended to competing risks analysis. The targeted audience includes biostatisticians, graduate students, and PhD (bio)statisticians. Knowledge in survival analysis at an introductory graduate level is advisable prior to reading this book.

  18. Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

    Science.gov (United States)

    Osteikoetxea, Xabier; Németh, Andrea; Sódar, Barbara W; Vukman, Krisztina V; Buzás, Edit Irén

    2016-06-01

    In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  19. Real-time RT-PCR analysis of human histidine decarboxylase, a new marker for several types of leukemia and cancer.

    Science.gov (United States)

    Melgarejo, Esther; Medina, Miguel Angel; Paz, José Carlos; Sánchez-Jiménez, Francisca; Urdiales, José Luis

    2006-01-01

    Histamine is involved in different physiological and pathological responses, such as immune response, gastric acid secretion or neurotransmission, as either angiogenesis or cancer. Histidine decarboxylase (HDC) catalyzes the formation of histamine from histidine. HDC has been suggested as a new marker for neuroendocrine differentiation, inflammatory pathologies and several leukemia and highly malignant forms of cancer, such as melanoma and small cell lung carcinoma. In the present work, we describe the use of Syber Green-based quantitative real-time RT-PCR to determine the expression of histidine decarboxylase in human cells and tissue. As an internal control, glyceraldehyde 3-phosphate dehydrogenase was also amplified. The linear dynamic range of the assay covered 4 orders of magnitude for HDC amplification. The detection limit was 0.1 ng of total RNA extracted from HMC-1 cells. This method is simple, rapid, sensitive, and quantitative, and allows for the specific identification of cells and tissue expressing HDC, stressing its potential diagnostic usefulness in malignancies in which HDC is described as a new marker.

  20. Two Independent Histidines One in Human Prolactin and One in Its Receptor Are Critical for pH-dependent Receptor Recognition and Activation

    Energy Technology Data Exchange (ETDEWEB)

    M Kulkarni; M Tettamanzi; J Murphy; C Keeler; D Myszka; N Chayen; E Lolis; M Hodsdon

    2011-12-31

    Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL-receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL-receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

  1. Integrated logic gate for fluorescence turn-on detection of histidine and cysteine based on Ag/Au bimetallic nanoclusters-Cu²⁺ ensemble.

    Science.gov (United States)

    Sun, Jian; Yang, Fan; Zhao, Dan; Chen, Chuanxia; Yang, Xiurong

    2015-04-01

    By means of employing 11-mercaptoundecanoic acid (11-MUA) as a reducing agent and protecting ligand, we present straightforward one-pot preparation of fluorescent Ag/Au bimetallic nanoclusters (namely AgAuNCs@11-MUA) from AgNO3 and HAuCl4 in alkaline aqueous solution at room temperature. It is found that the fluorescence of AgAuNCs@11-MUA has been selectively quenched by Cu(2+) ions, and the nonfluorescence off-state of the as-prepared AgAuNCs@11-MUA-Cu(2+) ensemble can be effectively switched on upon the addition of histidine and cysteine. By incorporating Ni(2+) ions and N-ethylmaleimide, this phenomenon is further exploited as an integrated logic gate and a specific fluorescence turn-on assay for selectively and sensitively sensing histidine and cysteine has been designed and established based on the original noncovalent AgAuNCs@11-MUA-Cu(2+) ensemble. Under the optimal conditions, histidine and cysteine can be detected in the concentration ranges of 0.25-9 and 0.25-7 μM; besides, the detection limits are found to be 87 and 111 nM (S/N = 3), respectively. Furthermore, we demonstrate that the proposed AgAuNCs@11-MUA-based fluorescent assay can be successfully utilized for biological fluids sample analysis.

  2. Investigations on the growth aspects and characterization of semiorganic nonlinear optical single crystals of L-histidine and its hydrochloride derivative.

    Science.gov (United States)

    Anandan, P; Arivanandhan, M; Hayakawa, Y; Babu, D Rajan; Jayavel, R; Ravi, G; Bhagavannarayana, G

    2014-01-01

    Semiorganic single crystals of l-histidine and l-histidine hydrochloride monohydrate have been obtained in a single solution prepared from the mixture of l-histidine and hydrochloric acid in 1:2M ratio. Growth aspects of the single crystals have been discussed along with characterization studies. Crystal system and lattice parameters have been identified by X-ray diffraction analyses. It has been observed that the grown crystals possess orthorhombic system but with different set of lattice parameters. Presence of various functional groups has been identified and formation of two different crystals has been confirmed by Fourier transform infrared spectral analyses and FT-Raman studies. Linear and nonlinear optical properties have been studied by UV-Vis spectral analyses and Kurtz-Perry powder technique respectively. The thermal stability of the grown crystals was determined by thermal analyses. From the characterization studies it is found that both the crystals are useful for second harmonic generation applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose.

    Science.gov (United States)

    McMahan, S A; Burgess, R R

    1996-04-05

    Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose. This compound has a biotin as one functional group and a nitrilotriacetic acid as the other. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.

  4. Amino acid-mediated 'turn-off/turn-on' nanozyme activity of gold nanoclusters for sensitive and selective detection of copper ions and histidine.

    Science.gov (United States)

    Liu, Yan; Ding, Ding; Zhen, Yuanlin; Guo, Rong

    2017-06-15

    Herein, we presented a facile strategy for highly sensitive and selective detection of both Cu2+ and histidine (His) by combining the peroxidase-like nanozyme activity of gold nanoclusters with amino acid's ambidentate nature. The peroxidase-like catalytic ability of histidine-Au nanoclusters (His-AuNCs) can be inhibited by the addition of Cu2+. The sensitivity of this probe to Cu2+ is significant with a linear range of 1-100nM, and a low detection limit of 0.1nM. More interestingly, His-AuNC/Cu2+ undergoes recovery of the activity upon exposure to free His, because His/Cu2+ complex is more stable due to the participation of the imidazole ring of His. The method displays a good selectivity toward histidine over all the other amino acids, with a wide linear relationship in the range of 20nM-2μM, and a low detection limit of 20nM. The feasibility of the probe for the rapid analysis of copper ion and His in human serum has been demonstrated with satisfactory results. With the merits of high sensitivity and selectivity, simplification, low cost, and visual readout with the naked eye, this novel 'turn-off/turn-on' sensing approach based on the amino acid's ambidentate nature is potentially applicable to metal ions, amino acids and peptides in biological and environmental areas. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A Perspective on Extracellular Vesicles Proteomics

    Directory of Open Access Journals (Sweden)

    Livia Rosa-Fernandes

    2017-11-01

    Full Text Available Increasing attention has been given to secreted extracellular vesicles (EVs in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  6. Versatile roles of extracellular vesicles in cancer

    Science.gov (United States)

    Kosaka, Nobuyoshi; Yoshioka, Yusuke; Fujita, Yu

    2016-01-01

    Numerous studies have shown that non–cell-autonomous regulation of cancer cells is an important aspect of tumorigenesis. Cancer cells need to communicate with stromal cells by humoral factors such as VEGF, FGFs, and Wnt in order to survive. Recently, extracellular vesicles (EVs) have also been shown to be involved in cell-cell communication between cancer cells and the surrounding microenvironment and to be important for the development of cancer. In addition, these EVs contain small noncoding RNAs, including microRNAs (miRNAs), which contribute to the malignancy of cancer cells. Here, we provide an overview of current research on EVs, especially miRNAs in EVs. We also propose strategies to treat cancers by targeting EVs around cancer cells. PMID:26974161

  7. Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

    Science.gov (United States)

    Kanada, Masamitsu; Bachmann, Michael H; Contag, Christopher H

    2016-02-01

    Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Extracellular matrix motion and early morphogenesis.

    Science.gov (United States)

    Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

    2016-06-15

    For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. © 2016. Published by The Company of Biologists Ltd.

  9. Isolation of Platelet-Derived Extracellular Vesicles.

    Science.gov (United States)

    Aatonen, Maria; Valkonen, Sami; Böing, Anita; Yuana, Yuana; Nieuwland, Rienk; Siljander, Pia

    2017-01-01

    Platelets participate in several physiological functions, including hemostasis, immunity, and development. Additionally, platelets play key roles in arterial thrombosis and cancer progression. Given this plethora of functions, there is a strong interest of the role of platelet-derived (extracellular) vesicles (PDEVs) as functional mediators and biomarkers. Moreover, the majority of the blood-borne EVs are thought to originate from either platelets or directly from the platelet precursor cells, the megakaryocytes, which reside in the bone marrow. To circumvent confusion, we use the term PDEVs for both platelet-derived and/or megakaryocyte-derived EVs. PDEVs can be isolated from blood or from isolated platelets after activation. In this chapter, we describe all commonly used PDEV isolation methods from blood and prepurified platelets.

  10. Physiology and pathology of extracellular vesicules

    Directory of Open Access Journals (Sweden)

    M. A. Panteleev

    2017-01-01

    Full Text Available This year marks the 50th anniversary of the first publication about blood plasma microparticles. Initially considered as cell fragments or “platelet dust”, extracellular vesicles currently attracted the attention of biochemists, biophysicists, physicians, pharmacists around the world. They are heterogeneous in structure and derived from many cell types, express different antigen and contain variety of biomolecules that determines wide range of biological activity, including procoagulant, regenerative, immunomodulating, and others. They play an important role in the pathophysiology of different diseases and conditions – from infarction, injuries and pregnancies to the “graft versus host” disease. The vesicles as medicaments and their carriers, as well as the drugs that affect them, are a rapidly developing field of research.

  11. Fragmentation of extracellular matrix by hypochlorous acid

    DEFF Research Database (Denmark)

    Woods, Alan A; Davies, Michael Jonathan

    2003-01-01

    of the MPO-derived oxidant hypochlorous acid (HOCl) with extracellular matrix from vascular smooth muscle cells and healthy pig arteries has been examined. HOCl is rapidly consumed by such matrix samples, with the formation of matrix-derived chloramines or chloramides. The yield of these intermediates...... increases with HOCl dose. These materials undergo a time- and temperature-dependent decay, which parallels the release of sugar and protein components from the treated matrix, consistent with these species being important intermediates. Matrix damage is enhanced by species that increase chloramine....../chloramide decomposition, with copper and iron ions being effective catalysts, and decreased by compounds which scavenge chloramines/chloramides, or species derived from them. The effect of such matrix modifications on cellular behaviour is poorly understood, though it is known that changes in matrix materials can have...

  12. Extracellular enzymes of Fusarium graminearum isolates

    Directory of Open Access Journals (Sweden)

    Gisele Eleonora Kikot

    2010-08-01

    Full Text Available Fusarium graminearum isolates from three different agroecological regions in Argentina were examined according to the production of different extracellular enzyme activities of potential biotechnological interest: pectinases (PGase: polygalacturonase and PMGase: polymethylgalacturonase, cellulase (CMCase: carboxymethylcellulase and hemicellulase (xylanase. The isolates were grown in minimum salt medium supplemented with 0.25% glucose, 0.125% citric pectin and 0.125% oat bran as carbon sources and/or enzyme inducers. PGase activity was detected early (after two days of incubation in all the cultures; it was found to be the highest for all the isolates. PMGase was high only for those isolates of the II region. CMCase and endoxylanase activities were particularly found at late stages (after four and seven days of incubation, respectively and the maximum values were lower than pectinase activities.

  13. RNA Sequencing Analysis of Salivary Extracellular RNA.

    Science.gov (United States)

    Majem, Blanca; Li, Feng; Sun, Jie; Wong, David T W

    2017-01-01

    Salivary biomarkers for disease detection, diagnostic and prognostic assessments have become increasingly well established in recent years. In this chapter we explain the current leading technology that has been used to characterize salivary non-coding RNAs (ncRNAs) from the extracellular RNA (exRNA) fraction: HiSeq from Illumina® platform for RNA sequencing. Therefore, the chapter is divided into two main sections regarding the type of the library constructed (small and long ncRNA libraries), from saliva collection, RNA extraction and quantification to cDNA library generation and corresponding QCs. Using these invaluable technical tools, one can identify thousands of ncRNA species in saliva. These methods indicate that salivary exRNA provides an efficient medium for biomarker discovery of oral and systemic diseases.

  14. Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine

    Science.gov (United States)

    Grolla, Ambra A; Travelli, Cristina

    2016-01-01

    Abstract In this review, we focus on the secreted form of nicotinamide phosphoribosyltransferase (NAMPT); extracellular NAMPT (eNAMPT), also known as pre‐B cell colony‐enhancing factor or visfatin. Although intracellular NAMPT is a key enzyme in controlling NAD metabolism, eNAMPT has been reported to function as a cytokine, with many roles in physiology and pathology. Circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer. Because cytokines produced in the tumour micro‐environment play an important role in cancer pathogenesis, in part by reprogramming cellular metabolism, future improvements in cancer immunotherapy will require a better understanding of the crosstalk between cytokine action and tumour biology. In this review, the knowledge of eNAMPT in cancer will be discussed, focusing on its immunometabolic function as a metabokine, its secretion, its mechanism of action and possible roles in the cancer micro‐environment. PMID:27128025

  15. Extracellular Matrix Molecules Facilitating Vascular Biointegration

    Directory of Open Access Journals (Sweden)

    Martin K.C. Ng

    2012-08-01

    Full Text Available All vascular implants, including stents, heart valves and graft materials exhibit suboptimal biocompatibility that significantly reduces their clinical efficacy. A range of biomolecules in the subendothelial space have been shown to play critical roles in local regulation of thrombosis, endothelial growth and smooth muscle cell proliferation, making these attractive candidates for modulation of vascular device biointegration. However, classically used biomaterial coatings, such as fibronectin and laminin, modulate only one of these components; enhancing endothelial cell attachment, but also activating platelets and triggering thrombosis. This review examines a subset of extracellular matrix molecules that have demonstrated multi-faceted vascular compatibility and accordingly are promising candidates to improve the biointegration of vascular biomaterials.

  16. Extracellular matrix component signaling in cancer

    DEFF Research Database (Denmark)

    Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

    2016-01-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization...... and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromols. are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin......, or basement membrane glycoproteins, but also in terms of matrix rigidity. This can regulate the release and subsequent biol. activity of matrix-bound growth factors, for example, transforming growth factor-β. In the environment of tumors, there may be changes in cell populations and their receptor profiles...

  17. A Perspective on Extracellular Vesicles Proteomics.

    Science.gov (United States)

    Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

    2017-01-01

    Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  18. Bioengineering Human Myocardium on Native Extracellular Matrix

    Science.gov (United States)

    Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

    2015-01-01

    Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable

  19. Identification of a receptor for extracellular renalase.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI in wild type (WT mice. Therefore, we sought to identity the receptor for extracellular renalase.RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

  20. In vitro toxicology studies of extracellular vesicles.

    Science.gov (United States)

    Maji, Sayantan; Yan, Irene K; Parasramka, Mansi; Mohankumar, Swathi; Matsuda, Akiko; Patel, Tushar

    2017-03-01

    Extracellular vesicles (EVs) are membrane-bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC-EV) or from bovine milk (BM-EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC-EVs nor BM-EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM-EVs but not with MSC-EVs. MSC-EVs did not have any significant effect on either spontaneous or collagen-induced platelet aggregation. In contrast, BM-EVs were noted to increase collagen-induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM-EV. Neither MSC-EVs nor BM-EVs induced HL-60 phagocytosis, although BM-EVs decreased zymosan-induced phagocytosis. Furthermore, neither MSC-EVs nor BM-EVs induced nitric oxide production. Unlike MSC-EVs, BM-EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC-EVs and BM-EVs that may reflect variations in techniques for EV isolation, EV content or cross-species differences. The safety of MSC-EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM-EVs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Extracellular polymeric substances play roles in extracellular electron transfer of Shewanella oneidensis MR-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, En-Hua; Christensen, Hans Erik Mølager

    It is well known that microorganism is surrounded by extracellular polymeric substances (EPS) which include polysaccharides, proteins, glycoproteins, nucleic acids, phospholipids, and humic acids. However, previous studies on microbial extracellular electron transfer (EET) are conducted on cells......, such as DNA, humic acids and some proteins, are electrochemically active or semiconductive. Herein, we report experimental evidences of EPS role on EET for Shewanella oneidensis MR-1. Atomic force microscopy clearly showed that the cell surface was cleaned and few EPS could be observed on MR-1 after...... without extracting EPS or cells collected from log stage or early-steady stage cultures with little EPS. Therefore, microbial cells are believed in contact directly with each other or electrode. Such attempt apparently ignored the role of EPS in microbial EET, even though many components of EPS...

  2. Activation of Bvg-Repressed Genes in Bordetella pertussis by RisA Requires Cross Talk from Noncooperonic Histidine Kinase RisK.

    Science.gov (United States)

    Chen, Qing; Ng, Victoria; Warfel, Jason M; Merkel, Tod J; Stibitz, Scott

    2017-11-15

    The two-component response regulator RisA, encoded by open reading frame BP3554 in the Bordetella pertussis Tohama I genomic sequence, is a known activator of vrg genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the bvgAS virulence regulon. Here we demonstrate that RisA is phosphorylated in vivo and that RisA phosphorylation is required for activation of vrg genes. An adjacent histidine kinase gene, risS , is truncated by frameshift mutation in B. pertussis but not in Bordetella bronchiseptica or Bordetella parapertussis Neither deletion of risS ' or bvgAS nor phenotypic modulation with MgSO 4 affected levels of phosphorylated RisA (RisA∼P) in B. pertussis However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the vrg genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisA D60E mutant, indicating that an active conformation of RisA, but not phosphorylation per se , is crucial for vrg activation. Interestingly, expression of vrg genes is still modulated by MgSO 4 in cells harboring the RisA D60E mutation, suggesting that the activated RisA senses additional signals to control vrg expression in response to environmental stimuli. IMPORTANCE In B. pertussis , the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally regulated vrg genes requires RisA and is also repressed by the Bvg-activated BvgR. RisA is an OmpR-like response regulator, but RisA phosphorylation was not expected because the gene for its presumed, cooperonic, histidine kinase is inactivated by mutation. In this study, we demonstrate phosphorylation of RisA in vivo by a noncooperonic histidine kinase. We also show that RisA phosphorylation is necessary but not

  3. Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

    OpenAIRE

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2009-01-01

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C...

  4. Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly

    OpenAIRE

    Pan, Shujuan; Wang, Ruoning; Zhou, Xi; Corvera, Joe; Kloc, Malgorzata; Sifers, Richard; Gallick, Gary E; Lin, Sue-Hwa; Kuang, Jian

    2008-01-01

    Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to e...

  5. Extracellular vesicles are integral and functional components of the extracellular matrix.

    Science.gov (United States)

    Rilla, Kirsi; Mustonen, Anne-Mari; Arasu, Uma Thanigai; Härkönen, Kai; Matilainen, Johanna; Nieminen, Petteri

    2017-10-21

    Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  6. Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly.

    Science.gov (United States)

    Pan, Shujuan; Wang, Ruoning; Zhou, Xi; Corvera, Joe; Kloc, Malgorzata; Sifers, Richard; Gallick, Gary E; Lin, Sue-Hwa; Kuang, Jian

    2008-08-06

    Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in alpha5beta1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes alpha5beta1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605-709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.

  7. Production of extracellular laccase from the newly isolated Bacillus ...

    African Journals Online (AJOL)

    This study was carried out with aim of screening for extracellular thermostable laccase producing bacteria. Twenty-two (22) laccase positive strains were isolated from the selected environmental samples while extracellular laccase activity was detected only in six strains namely TM1, TMT1, PK4, PS1, TMS1 and ASP3.

  8. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    Science.gov (United States)

    Erdbrügger, Uta; Le, Thu H

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. Copyright © 2016 by the American Society of Nephrology.

  9. The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles ? about microvesicles, exosomes, ectosomes and other extracellular vesicles

    OpenAIRE

    L?tvall, Jan; Rajendran, Lawrence; Gho, Yong-Song; Thery, Clotilde; Wauben, Marca; Raposo, Graca; Sj?strand, Margareta; Taylor, Douglas; Telemo, Esbj?rn; Breakefield, Xandra O.

    2012-01-01

    In 2011, researchers around the world interested in extracellular vesicles (EV) joined forces and founded the International Society for Extracellular Vesicles (ISEV). Membership has grown to approximately 750 in eight months, and the Society’s first meeting will take place in Gothenburg, Sweden, on 18-21 April 2012. Already approximately 500 participants have been attracted to this event. These are signs of rapid expansion in global research in the field of EV.(Published: 16 April 2012)Citati...

  10. Gram-negative and Gram-positive bacterial extracellular vesicles.

    Science.gov (United States)

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Extracellular vesicles as new pharmacological targets to treat atherosclerosis.

    Science.gov (United States)

    Yin, Min; Loyer, Xavier; Boulanger, Chantal M

    2015-09-15

    Extracellular vesicles released by most cell types, include apoptotic bodies (ABs), microvesicles (MVs) and exosomes. They play a crucial role in physiology and pathology, contributing to "cell-to-cell" communication by modifying the phenotype and the function of target cells. Thus, extracellular vesicles participate in the key processes of atherosclerosis from endothelial dysfunction, vascular wall inflammation to vascular remodeling. The purpose of this review is to summarize recent findings on extracellular vesicle formation, structure, release and clearance. We focus on the deleterious and beneficial effects of extracellular vesicles in the development of atherosclerosis. The potential role of extracellular vesicles as biomarkers and pharmacological targets, their innate therapeutic capacity, or their use for novel drug delivery devices in atherosclerotic cardiovascular diseases will also be discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Nitrogen availability of biogas residues

    Energy Technology Data Exchange (ETDEWEB)

    El-Sayed Fouda, Sara

    2011-09-07

    The objectives of this study were to characterize biogas residues either unseparated or separated into a liquid and a solid phase from the fermentation of different substrates with respect to their N and C content. In addition, short and long term effects of the application of these biogas residues on the N availability and N utilization by ryegrass was investigated. It is concluded that unseparated or liquid separated biogas residues provide N at least corresponding to their ammonium content and that after the first fertilizer application the C{sub org}:N{sub org} ratio of the biogas residues was a crucial factor for the N availability. After long term application, the organic N accumulated in the soil leads to an increased release of N.

  13. In Vivo Neuroprotective Effect of Histidine-Tryptophan-Ketoglutarate Solution in an Ischemia/Reperfusion Spinal Cord Injury Animal Model

    Directory of Open Access Journals (Sweden)

    Shin Kwang Kang

    2016-08-01

    Full Text Available Background: Paraplegia is a devastating complication following operations on the thoracoabdominal aorta. We investigated whether histidine-tryptophan-ketoglutarate (HTK solution could reduce the extent of ischemia/reperfusion (IR spinal cord injuries in a rat model using a direct delivery method. Methods: Twenty-four Sprague-Dawley male rats were randomly divided into four groups. The sham group (n=6 underwent a sham operation, the IR group (n=6 underwent only an aortic occlusion, the saline infusion group (saline group, n=6 underwent an aortic occlusion and direct infusion of cold saline into the occluded aortic segment, and the HTK infusion group (HTK group, n=6 underwent an aortic occlusion and direct infusion of cold HTK solution into the occluded aortic segment. An IR spinal cord injury was induced by transabdominal clamping of the aorta distally to the left renal artery and proximally to the aortic bifurcation for 60 minutes. A neurological evaluation of locomotor function was performed using the modified Tarlov score after 48 hours of reperfusion. The spinal cord was harvested for histopathological and immunohistochemical examinations. Results: The spinal cord IR model using direct drug delivery in rats was highly reproducible. The Tarlov score was 4.0 in the sham group, 1.17±0.75 in the IR group, 1.33±1.03 in the saline group, and 2.67±0.81 in the HTK group (p=0.04. The histopathological analysis of the HTK group showed reduced neuronal cell death. Conclusion: Direct infusion of cold HTK solution into the occluded aortic segment may reduce the extent of spinal cord injuries in an IR model in rats.

  14. Sensitive detection of biothiols and histidine based on the recovered fluorescence of the carbon quantum dots–Hg(II) system

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Juan; Zhang, Fengshuang; Yan, Xu; Wang, Long; Yan, Jin [College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Ding, Hong [State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun 130012 (China); Ding, Lan, E-mail: dinglan@jlu.edu.cn [College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012 (China)

    2015-02-15

    Highlights: • Carbon quantum dots-based probe was used for detection of GSH, Cys or His. • The fluorescence of CQDs was quenched by Hg(II) and then recovered by GSH, Cys or His. • No further surface modification or purification of CQDs was required. • This sensor exhibits superior accuracy and sensitivity. • The proposed method was simple in design, fast in operation. - Abstract: In this paper, we presented a novel, rapid and highly sensitive sensor for glutathione (GSH), cysteine (Cys) and histidine (His) based on the recovered fluorescence of the carbon quantum dots (CQDs)–Hg(II) system. The CQDs were synthesized by microwave-assisted approach in one pot according to our previous report. The fluorescence of CQDs could be quenched in the presence of Hg(II) due to the coordination occurring between Hg(II) and functional groups on the surface of CQDs. Subsequently, the fluorescence of the CQDs–Hg(II) system was recovered gradually with the addition of GSH, Cys or His due to their stronger affinity with Hg(II). A good linear relationship was obtained from 0.10 to 20 μmol L{sup −1} for GSH, from 0.20 to 45 μmol L{sup −1} for Cys and from 0.50 to 60 μmol L{sup −1} for His, respectively. This method has been successfully applied to the trace detection of GSH, Cys or His in human serum samples with satisfactory results. The proposed method was simple in design and fast in operation, which demonstrated great potential in bio-sensing fields.

  15. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    Science.gov (United States)

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

  16. Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation.

    Science.gov (United States)

    Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S; Tang, Hui; McGurren, Kelly A; van Wijnen, Andre J; Lerman, Amir; Lerman, Lilach O

    2017-07-01

    Mesenchymal stem/stromal cells (MSCs) have distinct capability for renal repair, but may have safety concerns. MSC-derived extracellular vesicles emerged as a novel noncellular alternative. Using a porcine model of metabolic syndrome and renal artery stenosis we tested whether extracellular vesicles attenuate renal inflammation, and if this capacity is mediated by their cargo of the anti-inflammatory cytokine interleukin (IL) 10. Pigs with metabolic syndrome were studied after 16 weeks of renal artery stenosis untreated or treated four weeks earlier with a single intrarenal delivery of extracellular vesicles harvested from adipose tissue-derived autologous MSCs. Lean and sham metabolic syndrome animals served as controls (seven each). Five additional pigs with metabolic syndrome and renal artery stenosis received extracellular vesicles with pre-silenced IL10 (IL10 knock-down). Single-kidney renal blood flow, glomerular filtration rate, and oxygenation were studied in vivo and renal injury pathways ex vivo. Retention of extracellular vesicles in the stenotic kidney peaked two days after delivery and decreased thereafter. Four weeks after injection, extracellular vesicle fragments colocalized with stenotic-kidney tubular cells and macrophages, indicating internalization or fusion. Extracellular vesicle delivery attenuated renal inflammation, and improved medullary oxygenation and fibrosis. Renal blood flow and glomerular filtration rate fell in metabolic syndrome and renal artery stenosis compared to metabolic syndrome, but was restored in pigs treated with extracellular vesicles. These renoprotective effects were blunted in pigs treated with IL10-depleted extracellular vesicles. Thus, extracellular vesicle-based regenerative strategies might be useful for patients with metabolic syndrome and renal artery stenosis. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Extracellular ATP Induces Calcium Signaling in Odontoblasts.

    Science.gov (United States)

    Lee, B M; Jo, H; Park, G; Kim, Y H; Park, C K; Jung, S J; Chung, G; Oh, S B

    2017-02-01

    Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca(2+) concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca(2+)-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X

  18. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  19. Extracellular Vesicles and Autophagy in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Tianyang Gao

    2016-01-01

    Full Text Available Osteoarthritis (OA is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies.

  20. Extracellular ice phase transitions in insects.

    Science.gov (United States)

    Hawes, T C

    2014-01-01

    At temperatures below their temperature of crystallization (Tc), the extracellular body fluids of insects undergo a phase transition from liquid to solid. Insects that survive the transition to equilibrium (complete freezing of the body fluids) are designated as freeze tolerant. Although this phenomenon has been reported and described in many Insecta, current nomenclature and theory does not clearly delineate between the process of transition (freezing) and the final solid phase itself (the frozen state). Thus freeze tolerant insects are currently, by convention, described in terms of the temperature at which the crystallization of their body fluids is initiated, Tc. In fact, the correct descriptor for insects that tolerate freezing is the temperature of equilibrium freezing, Tef. The process of freezing is itself a separate physical event with unique physiological stresses that are associated with ice growth. Correspondingly there are a number of insects whose physiological cryo-limits are very specifically delineated by this transitional envelope. The distinction also has considerable significance for our understanding of insect cryobiology: firstly, because the ability to manage endogenous ice growth is a fundamental segregator of cryotype; and secondly, because our understanding of internal ice management is still largely nascent.

  1. EXTRACELLULAR POLYSACCHARIDES OF POTATO RING ROT PATHOGEN

    Directory of Open Access Journals (Sweden)

    Shafikova Т.N.

    2006-03-01

    Full Text Available Many bacteria, including phytopathogenic ones produce extracellular polysaccharides or exopolysaccharides which are universal molecules. Causal agent of potato ring rot, Clavibacter michiganensis subspecies sepedonicus, secretes exopolysaccharides which role in pathogenesis is poorly investigated. The aim of our research is to ascertain the composition and structure of Clavibacter michiganensis subspecies sepedonicus exopolysaccharides. Exopolysaccharides of Clavibacter michiganensis subspecies sepedonicus are determined to consist of 4-6 anionic and neutral components which have molecular weights from 700 kDa. Glucose is a major monomer of polysaccharides and arabinose, rhamnose and mannose are minor monomers. Glucose is present in α-Dglucopyranose and β-D-glucopyranose configurations. Calcium is determined to be a component of exopolysaccharides. Components of exopolysaccharides of potato ring rot pathogen are probably capableto associate via calcium ions and other ionic interactions that may result in a change of their physiological activity. Further studies of Clavibacter michiganensis subspecies sepedonicus exopolysaccharides composition and structure can serve a base for the synthesis of their chemical analogues with elicitor action.

  2. The NIH Extracellular RNA Communication Consortium

    Directory of Open Access Journals (Sweden)

    Alexandra M. Ainsztein

    2015-08-01

    Full Text Available The Extracellular RNA (exRNA Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies, (c identifying exRNA biomarkers of disease, (d demonstrating clinical utility of exRNAs as therapeutic agents and developing scalable technologies required for these studies, and (e developing a community resource, the exRNA Atlas, to provide the scientific community access to exRNA data, standardized exRNA protocols, and other useful tools and technologies generated by funded investigators.

  3. Vascular Extracellular Matrix and Arterial Mechanics

    Science.gov (United States)

    WAGENSEIL, JESSICA E.; MECHAM, ROBERT P.

    2009-01-01

    An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments. PMID:19584318

  4. Micro- and macrorheology of jellyfish extracellular matrix.

    Science.gov (United States)

    Gambini, Camille; Abou, Bérengère; Ponton, Alain; Cornelissen, Annemiek J M

    2012-01-04

    Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Extracellular and Intracellular Regulation of Calcium Homeostasis

    Directory of Open Access Journals (Sweden)

    Felix Bronner

    2001-01-01

    Full Text Available An organism with an internal skeleton must accumulate calcium while maintaining body fluids at a well-regulated, constant calcium concentration. Neither calcium absorption nor excretion plays a significant regulatory role. Instead, isoionic calcium uptake and release by bone surfaces causes plasma calcium to be well regulated. Very rapid shape changes of osteoblasts and osteoclasts, in response to hormonal signals, modulate the available bone surfaces so that plasma calcium can increase when more low-affinity bone calcium binding sites are made available and can decrease when more high-affinity binding sites are exposed. The intracellular free calcium concentration of body cells is also regulated, but because cells are bathed by fluids with vastly higher calcium concentration, their major regulatory mechanism is severe entry restriction. All cells have a calcium-sensing receptor that modulates cell function via its response to extracellular calcium. In duodenal cells, the apical calcium entry structure functions as both transporter and a vitamin D–responsive channel. The channel upregulates calcium entry, with intracellular transport mediated by the mobile, vitamin D–dependent buffer, calbindin D9K, which binds and transports more than 90% of the transcellular calcium flux. Fixed intracellular calcium binding sites can, like the body's skeleton, take up and release calcium that has entered the cell, but the principal regulatory tool of the cell is restricted entry.

  6. Extracellular DNA Contributes to Dental Biofilm Stability.

    Science.gov (United States)

    Schlafer, Sebastian; Meyer, Rikke L; Dige, Irene; Regina, Viduthalai R

    2017-01-01

    Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control. © 2017 S. Karger AG, Basel.

  7. Extracellular DNA Contributes to Dental Biofilm Stability

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise; Dige, Irene

    2017-01-01

    dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated...... the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover......Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown...

  8. Extracellular Vesicles and Autophagy in Osteoarthritis

    Science.gov (United States)

    Guo, Weimin; Chen, Mingxue; Huang, Jingxiang; Yuan, Zhiguo; Zhang, Yu; Wang, Mingjie; Li, Penghao; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Zhang, Li; Xu, Wenjing; Lu, Shibi

    2016-01-01

    Osteoarthritis (OA) is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs) are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies. PMID:28078284

  9. Bacterial Extracellular Polysaccharides Involved in Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Elena P. Ivanova

    2009-07-01

    Full Text Available Extracellular polymeric substances (EPS produced by microorganisms are a complex mixture of biopolymers primarily consisting of polysaccharides, as well as proteins, nucleic acids, lipids and humic substances. EPS make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix. The key functions of EPS comprise the mediation of the initial attachment of cells to different substrata and protection against environmental stress and dehydration. The aim of this review is to present a summary of the current status of the research into the role of EPS in bacterial attachment followed by biofilm formation. The latter has a profound impact on an array of biomedical, biotechnology and industrial fields including pharmaceutical and surgical applications, food engineering, bioremediation and biohydrometallurgy. The diverse structural variations of EPS produced by bacteria of different taxonomic lineages, together with examples of biotechnological applications, are discussed. Finally, a range of novel techniques that can be used in studies involving biofilm-specific polysaccharides is discussed.

  10. Routes and mechanisms of extracellular vesicle uptake

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    Laura Ann Mulcahy

    2014-08-01

    Full Text Available Extracellular vesicles (EVs are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.

  11. Functional transferred DNA within extracellular vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Jin [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province (China); Wu, Gengze [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Jose, Pedro A. [Division of Nephrology, Department of Medicine and Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 (United States); Zeng, Chunyu, E-mail: Chunyuzeng01@163.com [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China)

    2016-11-15

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  12. Mycoplasma bovis escapes bovine neutrophil extracellular traps.

    Science.gov (United States)

    Gondaira, Satoshi; Higuchi, Hidetoshi; Nishi, Koji; Iwano, Hidetomo; Nagahata, Hajime

    2017-02-01

    Mycoplasma bovis is a significant pathogen in bovine infections including mastitis, pneumonia, arthritis and otitis media, and is the cause of large economic losses in beef and dairy farms. During infection with M. bovis, recruited neutrophils are not sufficient to eradicate M. bovis from the infection site. The release of neutrophil extracellular traps (NETs) is one of the innate immune responses of neutrophils but the effect of M. bovis on NET formation by bovine neutrophils has not yet been clarified. The objective of our research was to examine the effect of M. bovis on NET formation and the killing activity of bovine neutrophils. We showed that NETs were not detected following stimulation of neutrophils by M. bovis alone or with Phorbol 12-myristate 13-acetat (PMA). Reactive oxygen species production is essential for NET formation but the levels in neutrophils stimulated with M. bovis at multiplicity of infections of 10, 100, and 1000 were similar to those of unstimulated cells. NET formation induced by PMA stimulated neutrophils disappeared following the addition of M. bovis but this phenomenon was not observed when ethylenediaminetetraacetic acid (EDTA) was added. M. bovis colony forming units were significantly decreased by the addition of EDTA in the presence of NETs. Our results suggested that M. bovis infection alone did not induce NETs and that M. bovis nucleases, as hypothesis-based, contributed to resistance against the killing activity of NETs. Copyright © 2016. Published by Elsevier B.V.

  13. Towards traceable size determination of extracellular vesicles

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    Zoltán Varga

    2014-02-01

    Full Text Available Background: Extracellular vesicles (EVs have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. Methods: In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS and size exclusion chromatography coupled with dynamic light scattering detection. Results: The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. Conclusion: SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required.

  14. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    Science.gov (United States)

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  15. Evaluation of residue-residue contact prediction in CASP10

    KAUST Repository

    Monastyrskyy, Bohdan

    2013-08-31

    We present the results of the assessment of the intramolecular residue-residue contact predictions from 26 prediction groups participating in the 10th round of the CASP experiment. The most recently developed direct coupling analysis methods did not take part in the experiment likely because they require a very deep sequence alignment not available for any of the 114 CASP10 targets. The performance of contact prediction methods was evaluated with the measures used in previous CASPs (i.e., prediction accuracy and the difference between the distribution of the predicted contacts and that of all pairs of residues in the target protein), as well as new measures, such as the Matthews correlation coefficient, the area under the precision-recall curve and the ranks of the first correctly and incorrectly predicted contact. We also evaluated the ability to detect interdomain contacts and tested whether the difficulty of predicting contacts depends upon the protein length and the depth of the family sequence alignment. The analyses were carried out on the target domains for which structural homologs did not exist or were difficult to identify. The evaluation was performed for all types of contacts (short, medium, and long-range), with emphasis placed on long-range contacts, i.e. those involving residues separated by at least 24 residues along the sequence. The assessment suggests that the best CASP10 contact prediction methods perform at approximately the same level, and comparably to those participating in CASP9.

  16. Dissection of Functional Residues in Receptor Activity-Modifying Proteins Through Phylogenetic and Statistical Analyses

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    Alfonso Benítez-Páez

    2008-01-01

    Full Text Available Type I and type-II functional divergences have been stated to highlight specific residues carrying out differential functions in evolutionary-divergent protein clusters from a single common ancestor. Briefly, type I analysis is based on residue constraints reflecting a gain of function just in one cluster of an entire family of proteins; while the type-II approach is based on residue constraints showing a different chemical nature in every cluster of a protein family. This last evidence is understood as differential functionality among clusters. The Receptor Activity-Modifying Proteins constitute a family characterized by its paralogous distribution in vertebrates. They are known as G-Protein Coupled Receptor modulators. Although several studies have determined their involvement in ligand binding, specificity, and enhancement of signal transduction, the responsible residues supporting those functions are unclear. Using different bioinformatic approaches, we predicted residues involved in different RAMP functional tasks. Many residues localized in an extracellular coil of RAMP proteins were predicted to be under functional divergence suggesting a gain of function in their respective proteins. Interestingly, the transmembrane region also showed important results for residues playing relevant roles where most of them showed a biased distribution on the structure. A relevant role was conferred by the enrichment of type-II residues observed in their sequences. We show a collection of residues explaining possible gain of function and differential functionality in RAMP proteins. These residues are still experimentally unexplored with regards to functionality. Finally, an evolutionary history could be discerned. Mainly, the RAMP2 cluster has evolved in a higher manner than other RAMP clusters. However, a deacceleration in the aminoacid substitution rate of RAMP2 was observed in mammals. Such effect could be caused by the co-evolution of ligands and

  17. Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382

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    Jelle B. Bultema

    2014-01-01

    Full Text Available The Bacillus circulans ATCC 31382 β-galactosidase (BgaD is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2. Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS. The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447 and nucleophile (E532, and as transition state stabilizers (H345, H379, respectively.

  18. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

    Directory of Open Access Journals (Sweden)

    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  19. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

    Science.gov (United States)

    Pinto, Ana F; Romão, Célia V; Pinto, Liliana C; Huber, Harald; Saraiva, Lígia M; Todorovic, Smilja; Cabelli, Diane; Teixeira, Miguel

    2015-01-01

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  20. The Catalase Activity of Catalase-Peroxidases Is Modulated by Changes in the pKa of the Distal Histidine.

    Science.gov (United States)

    Machuqueiro, Miguel; Victor, Bruno; Switala, Jacek; Villanueva, Jacylyn; Rovira, Carme; Fita, Ignacio; Loewen, Peter C

    2017-05-02

    The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu ∼7 and ∼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKa of the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct.

  1. Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species.

    Science.gov (United States)

    Notermans, S; Wieten, G; Engel, H W; Rombouts, F M; Hoogerhout, P; van Boom, J H

    1987-02-01

    Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.

  2. Prediction of extracellular matrix proteins based on distinctive sequence and domain characteristics.

    Science.gov (United States)

    Jung, Juhyun; Ryu, Taewoo; Hwang, Yongdeuk; Lee, Eunjung; Lee, Doheon

    2010-01-01

    Extracellular matrix (ECM) proteins are secreted to the exterior of the cell, and function as mediators between resident cells and the external environment. These proteins not only support cellular structure but also participate in diverse processes, including growth, hormonal response, homeostasis, and disease progression. Despite their importance, current knowledge of the number and functions of ECM proteins is limited. Here, we propose a computational method to predict ECM proteins. Specific features, such as ECM domain score and repetitive residues, were utilized for prediction. Based on previously employed and newly generated features, discriminatory characteristics for ECM protein categorization were determined, which significantly improved the performance of Random Forest and support vector machine (SVM) classification. We additionally predicted novel ECM proteins from non-annotated human proteins, validated with gene ontology and earlier literature. Our novel prediction method is available at biosoft.kaist.ac.kr/ecm.

  3. Extracellular vesicles are the Trojan horses of viral infection.

    Science.gov (United States)

    Altan-Bonnet, Nihal

    2016-08-01

    Extracellular vesicles have recently emerged as a novel mode of viral propagation exploited by both enveloped and non-enveloped viruses. In particular non-enveloped viruses utilize the hosts' production of extracellular vesicles to exit from cells non-lytically and to hide and manipulate the immune system. Moreover, challenging the long held idea that viruses behave as independent genetic units, extracellular vesicles enable multiple viral particles and genomes to collectively traffic in and out of cells, which can promote genetic cooperativity among viral quasispecies and enhance the fitness of the overall viral population. Published by Elsevier Ltd.

  4. Extracellular electron transfer mechanisms between microorganisms and minerals

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Liang; Dong, Hailiang; Reguera, Gemma; Beyenal, Haluk; Lu, Anhuai; Liu, Juan; Yu, Han-Qing; Fredrickson, James K.

    2016-08-30

    Electrons can be transferred from microorganisms to multivalent metal ions that are associated with minerals and vice versa. As the microbial cell envelope is neither physically permeable to minerals nor electrically conductive, microorganisms have evolved strategies to exchange electrons with extracellular minerals. In this Review, we discuss the molecular mechanisms that underlie the ability of microorganisms to exchange electrons, such as c-type cytochromes and microbial nanowires, with extracellular minerals and with microorganisms of the same or different species. Microorganisms that have extracellular electron transfer capability can be used for biotechnological applications, including bioremediation, biomining and the production of biofuels and nanomaterials.

  5. Extracellular matrix components as therapeutics for spinal cord injury.

    Science.gov (United States)

    Haggerty, Agnes E; Marlow, Megan M; Oudega, Martin

    2017-06-23

    There is no treatment for people with spinal cord injury that leads to significant functional improvements. The extracellular matrix is an intricate, 3-dimensional, structural framework that defines the environment for cells in the central nervous system. The components of extracellular matrix have signaling and regulatory roles in the fate and function of neuronal and non-neuronal cells in the central nervous system. This review discusses the therapeutic potential of extracellular matrix components for spinal cord repair. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity.

    Science.gov (United States)

    Lynagh, Timothy; Khamu, Tina S; Bryan-Lluka, Lesley J

    2014-01-01

    The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [(3)H]noradrenaline uptake, [(3)H]nisoxetine binding and [(3)H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [(3)H]nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [(3)H]noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [(3)H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [(3)H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT.

  7. Efficient decellularization for bovine pericardium with extracellular matrix preservation and good biocompatibility.

    Science.gov (United States)

    Li, Ning; Li, Yang; Gong, Dejun; Xia, Cuiping; Liu, Xiaohong; Xu, Zhiyun

    2018-01-10

    In this study, we sought to explore an efficient decellularization protocol for bovine pericardia with better extracellular matrix preservation and good biocompatibility. Bovine pericardia were decellularized by sodium dodecyl sulphate (SDS), SDS + sodium deoxycholate (SD), Triton X-100 (TX), TX + SD (TS), freeze-thaw cycles + SDS + SD (FSS) and freeze-thaw cycles + TX + SD (FTS), respectively. Untreated pericardia were used as native control. Histological examination, residual cellular content analysis, biochemical and biomechanical evaluations and cytotoxicity assay were performed to investigate decellularization efficiency, xenoantigens removal, extracellular matrix preservation and biocompatibility. In vivo biocompatibility was evaluated using a subcutaneous implantation method in rats. Among these protocols, FSS and FTS protocols were the most effective methods to remove both the DNA material and the galactose-α-1,3-galactose antigen. TX, TS and FTS bovine pericardia maintained the collagen content and had no cytotoxicity to human umbilical vein endothelial cells. The contents of elastin and glycosaminoglycan were lost to different degrees after decellularization, with the highest content of preservation with TX, followed by TS and FTS. Consistently, no significant difference was found between native bovine pericardia and TX, TS or FTS bovine pericardia. In vivo, FTS implants had minimal infiltration of macrophages and T-lymphocytes, with no histological evidence of peri-implant necrosis and calcification. These results suggested that the FTS protocol showed optimal decellularization results with better extracellular matrix preservation and good biocompatibility. It may be a suitable protocol for producing a suitable scaffold for heart tissue engineering.

  8. Suppression of human solid tumor growth in mice by intratumor and systemic inoculation of histidine-rich and pH-dependent host defense-like lytic peptides.

    Science.gov (United States)

    Makovitzki, Arik; Fink, Avner; Shai, Yechiel

    2009-04-15

    Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and l-amino acids ([D]-K(6)L(9)) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, approximately 10.5) for histidines (pKa, approximately 6.1) in the parental peptide [D]-K(6)L(9). Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K(3)H(3)L(9) and [D]-H(6)L(9). Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H(6)L(9) mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors.

  9. Preparation and evaluation of poly(l-histidine based pH-sensitive micelles for intracellular delivery of doxorubicin against MCF-7/ADR cells

    Directory of Open Access Journals (Sweden)

    Nan Jia

    2017-09-01

    Full Text Available In this study, a pH-sensitive micelle self-assembled from poly(l-histidine based triblock copolymers of poly(ethylene glycol–poly(d,l-lactide–poly(l-histidine (mPEG-PLA-PHis was prepared and used as the intracellular doxorubicin (Dox delivery for cancer chemotherapy. Dox was loaded into the micelles by thin-film hydration method and a Box–Behnken design for three factors at three levels was used to optimize the preparations. The optimized mPEG-PLA-Phis/Dox micelles exhibited good encapsulation efficiency of 91.12%, a mean diameter of 45 nm and narrow size distribution with polydispersity index of 0.256. In vitro drug release studies demonstrated that Dox was released from the micelles in a pH-dependent manner. Furthermore, the cellular evaluation of Dox loaded micelles displayed that the micelles possessed high antitumor activity in vitro with an IC50 of 35.30 µg/ml against MCF-7/ADR cells. The confocal microscopy and flow cytometry experiments indicated that mPEG-PLA-Phis micelles mediated efficient cytoplasmic delivery of Dox with the aid of poly(l-histidine mediated endosomal escape. In addition, blank mPEG-PLA-Phis micelles were shown to be nontoxic to MCF-7/ADR cells even at a high concentration of 200 µg/ml. The pH-sensitive mPEG-PLA-PHis micelles have been demonstrated to be a promising nanosystem for the intracellular delivery of Dox for MDR reversal.

  10. Biological reference materials for extracellular vesicle studies.

    Science.gov (United States)

    Valkonen, S; van der Pol, E; Böing, A; Yuana, Y; Yliperttula, M; Nieuwland, R; Laitinen, S; Siljander, P R M

    2017-02-15

    Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  11. An immunoassay for urinary extracellular vesicles.

    Science.gov (United States)

    Salih, Mahdi; Fenton, Robert A; Knipscheer, Jeroen; Janssen, Joost W; Vredenbregt-van den Berg, Mirella S; Jenster, Guido; Zietse, Robert; Hoorn, Ewout J

    2016-04-15

    Although nanosized urinary extracellular vesicles (uEVs) are increasingly used for biomarker discovery, their isolation currently relies on time-consuming techniques hindering high-throughput application. To navigate this problem, we designed an immunoassay to isolate, quantify, and normalize uEV proteins. The uEV immunoassay consists of a biotinylated CD9 antibody to isolate uEVs, an antibody against the protein of interest, and two conjugated antibodies to quantify the protein of interest and CD9. As a proof of principle, the immunoassay was developed to analyze the water channel aquaporin-2 (AQP2) and the sodium-chloride cotransporter (NCC). CD9 was used as a capture antibody because immunoprecipitation showed that anti-CD9 antibody, but not anti-CD63 antibody, isolated AQP2 and NCC. CD9 correlated strongly with urine creatinine, allowing CD9 to be used for normalization of spot urines. The uEV immunoassay detected AQP2 and NCC with high sensitivity, low coefficients of variance, and stability in dilution series. After water loading in healthy subjects, the uEV immunoassay detected decreases in AQP2 and NCC equally well as the traditional method using ultracentrifugation and immunoblot. The uEV immunoassay also reliably detected lower and higher AQP2 or NCC levels in uEVs from patients with pathological water or salt reabsorption, respectively. In summary, we report a novel approach to analyze uEVs that circumvents existing isolation and normalization issues, requires small volumes of urine, and detects anticipated changes in physiological responses and clinical disorders. Copyright © 2016 the American Physiological Society.

  12. Procoagulant extracellular vesicles in amniotic fluid.

    Science.gov (United States)

    Hell, Lena; Wisgrill, Lukas; Ay, Cihan; Spittler, Andreas; Schwameis, Michael; Jilma, Bernd; Pabinger, Ingrid; Altevogt, Peter; Thaler, Johannes

    2017-06-01

    Embolization of amniotic fluid (AF) into the blood circulation leads to disseminated intravascular coagulation (DIC). Procoagulant phosphatidylserine (PS)- and tissue factor (TF)-exposing extracellular vesicles (EVs) might play an important role in AF embolism-induced DIC. It was the aim of the present study to perform analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry. We applied a prothrombinase assay (that quantifies PS exposure on EVs), an EV-associated TF activity assay, a fibrin generation assay, a thrombin generation assay, a whole blood clotting model, and flow cytometry in AF and control plasma. We found that PS exposure on EVs was 21-fold increased in AF compared with plasma. Also, EV-associated TF activity was highly increased in AF compared with plasma. AF-derived EVs activated the blood coagulation cascade via PS and TF in the fibrin and thrombin generation assays. In a whole blood clotting model, AF-derived EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor XII (FXII) was not affected. Applying flow cytometry, a subpopulation of PS+ and TF+ EVs was identified in AF but not in control plasma. In conclusion, we investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine their procoagulant potential. Taken together, our data further delineate the pathomechanisms underlying AF-induced coagulopathy. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Extracellular vesicles in obesity and diabetes mellitus.

    Science.gov (United States)

    Pardo, Fabián; Villalobos-Labra, Roberto; Sobrevia, Bastián; Toledo, Fernando; Sobrevia, Luis

    2017-11-24

    Cell-to-cell communication happens via diverse mechanisms including the synthesis, release and transfer to target cells of extracellular vesicles (EVs). EVs include nanovesicles (i.e., exosomes) and microvesicles, including apoptotic bodies. The amount and cargo of released EVs, which consist of microRNAs (miRNAs), mRNA, proteins, DNA, among other molecules, are altered in obesity and diabetes mellitus. EVs from these diseases show with altered cargo including several miRNAs and the enrichment with molecules involved in inflammation, immune efficiency, and cell activation. The role of EVs in obesity regards with adipocytes-released vesicles that may end in a systemic insulin resistance. In diabetes mellitus, the exosomes cargo may signal to transform a normal phenotype into a diabetic phenotype in endothelial cells. The evidence of EVs as modulators of cell function is increasing; however, it is still unclear whether exosomes or microvesicles are a trustable and useful marker for the diagnose or early detection of obesity or diabetes mellitus. In this review, we summarise the reported information regarding EVs involvement in obesity, T1 and T2 diabetes mellitus, and gestational diabetes mellitus. We emphasise the fact that studies addressing a potential effect of obesity or diabetes mellitus on cell function and the severity of the diseases are done in patients suffering simultaneously with both of these diseases, i.e., diabesity. Unfortunately, the lack of information regarding the biological effects and the potential involved mechanisms makes difficult to understand the role of the EVs as a marker of these and perhaps other diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Filtration recovery of extracellular DNA from environmental ...

    Science.gov (United States)

    qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular DNA (eDNA) that is co-recovered by the filtration procedure which is the most commonly used method to concentrate target microbes from environmental waters. Using C. parvum 18S rRNA gene fragment as a representative of eDNA, we examined the impact of filters (types and pore sizes) and physiochemical properties of surface water samples on the recovery of spiked DNA. Our results indicated that binding affinities of various filter membranes were quantifiably different for eDNA fragments with the polycarbonate (PC) binding the least and mixed cellulose acetate and cellulose nitrate (MCE) binding the most as evidenced by up to 16% recovery of the spiked plasmid DNA with a pore size of 0.2µm. Water quality parameters also had a distinct influence on the recovery of eDNA which was enhanced by the presence of high total suspended solid (TSS) concentrations and reduced pH. At pH 5.5, with 150mg/L of clay, DNA recovery was increased to as much as 18%. By shielding the negative charge, thus increasing the interaction of DNA and colloids, the increase of Na+ and Ca+2 concentrations resulted in more DNA binding and consequently more recovery from environmental water samples. Therefore, in addition

  15. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  16. Lung extracellular matrix and redox regulation.

    Science.gov (United States)

    Watson, Walter H; Ritzenthaler, Jeffrey D; Roman, Jesse

    2016-08-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an 'end-stage' process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation-reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  17. TRANSGLUTAMINASE-2: A NEW ENDOSTATIN PARTNER IN THE EXTRACELLULAR MATRIX OF ENDOTHELIAL CELLS

    Science.gov (United States)

    Faye, Clément; Inforzato, Antonio; Bignon, Marine; Hartmann, Daniel J.; Muller, Laurent; Ballut, Lionel; Olsen, Bjorn R.; Day, Anthony J.; Ricard-Blum, Sylvie

    2010-01-01

    Endostatin, the C-terminal domain of collagen XVIII, binds to transglutaminase-2 (TG-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (KD = 6.8 nM). Enzymatic assays indicated that, in contrast to other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, R27 and R139, are crucial for its binding to TG-2. They are also involved in the binding to heparin (Sasaki et al., EMBO J 18:6240–6248), and to α5β1 and αvβ3 integrins (Faye et al., J Biol Chem 284:22029–22040), suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the GTP binding site of TG-2 as a potential binding site for endostatin. Endostatin and TG-2 are colocalized in the extracellular matrix secreted by endothelial cells under hypoxia, that stimulates angiogenesis. This interaction occurring in a cellular context might participate in the concerted regulation of angiogenesis, and tumorigenesis by the two proteins. PMID:20156196

  18. Supramolecular Self-Assembly of Histidine-Capped-Dialkoxy-Anthracene: A Visible Light Triggered Platform for facile siRNA Delivery

    KAUST Repository

    Patil, Sachin

    2016-06-29

    Supramolecular self-assembly of histidine-capped-dialkoxy-anthracene (HDA) results in the formation of light responsive nanostructures.Single-crystal X-ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond is established between the imidazole moieties while the second involves the oxygen atom of one amide group and the hydrogen atom of a second amide group. When protonated in acidic aqueous media, HDA successfully complexes siRNA yielding spherical nanostructures. This biocompatible platform controllably delivers siRNA with high efficacy upon visible light irradiation leading up to 90% of gene silencing in live cells.

  19. Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways

    OpenAIRE

    Hsiao, Hui-Yi; He, Qingfang; van Waasbergen, Lorraine G.; Grossman, Arthur R.

    2004-01-01

    We have deleted a gene for a sensor histidine kinase, dspA (or hik33), in the cyanobacterium Synechocystis sp. strain PCC6803. In low and moderate light, the mutant grew slowly under photoautotrophic conditions, with a doubling time of ∼40 h, and had severely reduced photosynthetic oxygen evolution. When the mutant was maintained in low or moderate light in the presence of glucose, its growth rate was only somewhat lower than that of wild-type cells. However, the mutant was light sensitive an...

  20. catena-Poly[[bis(nitrato-κ2O,O′barium]-bis(μ-l-histidine-κ3O,O′:O

    Directory of Open Access Journals (Sweden)

    P. Arularasan

    2013-11-01

    Full Text Available In the polymeric title compound, [Ba(NO32(C6H9N3O22]n, the BaII atom is located on a crystallographic twofold axis and is coordinated by ten O atoms. Six are derived from two zwitterionic l-histidine molecules that simultaneously chelate one BaII atom and bridge to another. The remaining four O atoms are derived from two chelating nitrates. The molecules assemble to form a chain along [010]. In the crystal, chains are linked via N—H...O and N—H...N hydrogen bonds, generating a three-dimensional network.