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Sample records for acid residues essential

  1. Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Takahashi, T; Hiramoto, S; Wato, S; Nishimoto, T; Wada, Y; Nagai, K; Yamaguchi, H

    1999-11-01

    Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue. PMID:10544275

  2. Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

    PubMed

    Cheng, H F; Jiang, M J; Chen, C L; Liu, S M; Wong, L P; Lomasney, J W; King, K

    1995-03-10

    In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC. PMID:7890667

  3. Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin.

    PubMed

    Martin, Virginie; Groenendyk, Jody; Steiner, Simone S; Guo, Lei; Dabrowska, Monika; Parker, J M Robert; Müller-Esterl, Werner; Opas, Michal; Michalak, Marek

    2006-01-27

    Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor. PMID:16291754

  4. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

    PubMed

    Shiheido, Hirokazu; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. PMID:25600804

  5. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    SciTech Connect

    Shiheido, Hirokazu Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  6. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  7. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    PubMed Central

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  8. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    PubMed

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  9. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding. PMID:25239139

  10. Purification, Cloning, Characterization and Essential Amino Acid Residues Analysis of a New ι-Carrageenase from Cellulophaga sp. QY3

    PubMed Central

    Ma, Su; Duan, Gaofei; Chai, Wengang; Geng, Cunliang; Tan, Yulong; Wang, Lushan; Le Sourd, Frédéric; Michel, Gurvan; Yu, Wengong; Han, Feng

    2013-01-01

    ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0–10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites −1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites −1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites −1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82. PMID:23741363

  11. Discovery of essential fatty acids

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2015-01-01

    Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research. PMID:25339684

  12. AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport

    PubMed Central

    Wang, Liguang; Wu, Xuexia; Liu, Yafen; Qiu, Quan-Sheng

    2015-01-01

    AtNHX5 and AtNHX6, the endosomal Na+,K+/H+ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K+ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K+ and H+ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K+ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K+ at acidic pH while AtCHXs at low K+ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K+ and was sensitive to low K+ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K+ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K+ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K+ transport. PMID:26650539

  13. Identification of essential residues for the catalytic function of 85-kDa cytosolic phospholipase A2. Probing the role of histidine, aspartic acid, cysteine, and arginine.

    PubMed

    Pickard, R T; Chiou, X G; Strifler, B A; DeFelippis, M R; Hyslop, P A; Tebbe, A L; Yee, Y K; Reynolds, L J; Dennis, E A; Kramer, R M; Sharp, J D

    1996-08-01

    Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel. PMID:8702602

  14. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    PubMed

    Tay, Moon Y F; Smith, Kate; Ng, Ivan H W; Chan, Kitti W K; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Luo, Dahai; Jans, David A; Forwood, Jade K; Vasudevan, Subhash G

    2016-09-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  15. Identification of amino-acid residues in the V protein of peste des petits ruminants essential for interference and suppression of STAT-mediated interferon signaling.

    PubMed

    Ma, Xusheng; Yang, Xing; Nian, Xiaofeng; Zhang, Zhidong; Dou, Yongxi; Zhang, Xuehu; Luo, Xuenong; Su, Junhong; Zhu, Qiyun; Cai, Xuepeng

    2015-09-01

    Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. PMID:25965795

  16. Identification of amino-acid residues in the V protein of peste des petits ruminants essential for interference and suppression of STAT-mediated interferon signaling

    SciTech Connect

    Ma, Xusheng; Yang, Xing; Nian, Xiaofeng; Zhang, Zhidong; Dou, Yongxi; Zhang, Xuehu; Luo, Xuenong; Su, Junhong; Zhu, Qiyun; Cai, Xuepeng

    2015-09-15

    Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.

  17. Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

    PubMed

    Riemer, D; Dodemont, H; Weber, K

    1991-12-01

    We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID:1724961

  18. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  19. Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.

    PubMed Central

    Tsumori, H; Minami, T; Kuramitsu, H K

    1997-01-01

    A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes. PMID:9171379

  20. Essential Fatty Acids as Transdermal Penetration Enhancers.

    PubMed

    van Zyl, Lindi; du Preez, Jan; Gerber, Minja; du Plessis, Jeanetta; Viljoen, Joe

    2016-01-01

    The aim of this study was to investigate the effect of different penetration enhancers, containing essential fatty acids (EFAs), on the transdermal delivery of flurbiprofen. Evening primrose oil (EPO), vitamin F, and Pheroid technology all contain fatty acids and were compared using a cream-based formulation. This selection was to ascertain whether EFAs solely, or EFAs in a Pheroid delivery system, would have a significant increase in the transdermal delivery of a compound. Membrane release studies were performed, and the results indicated the following rank order for flurbiprofen release from the different formulations: vitamin F > control > EPO > Pheroid. Topical skin delivery results indicated that flurbiprofen was present in the stratum corneum-epidermis and the epidermis-dermis. The average percentage flurbiprofen diffused to the receptor phase (representing human blood) indicated that the EPO formulation showed the highest average percentage diffused. The Pheroid formulation delivered the lowest concentration with a statistical significant difference (p < 0.05) compared with the control formulation (containing 1% flurbiprofen and no penetration enhancers). The control formulation presented the highest average flux, with the EPO formulation following the closest. It could, thus, be concluded that EPO is the most favorable chemical penetration enhancer when used in this formulation. PMID:26852854

  1. Bioconverted Products of Essential Fatty Acids as Potential Antimicrobial Agents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This review deals with the recent findings on the microbial conversion of essential fatty acids (EFAs) through Pseudomonas aeruginosa PR3 NRRL-B-18602, and the antimicrobial properties of bioconverted essential fatty acids, with particular emphasis on n-3 or n-6 fatty acids. The first section deals...

  2. Essential fatty acid consumption and risk of breast cancer.

    PubMed

    Godley, P A

    1995-07-01

    Animal and ecological studies of essential fatty acids suggest that omega-3 fatty acids found in fish oils and omega-6 fatty acids found in vegetable oils may be playing a role in the etiology of breast cancer. Essential fatty acids may modulate breast cancer risk by interacting with prostaglandins, which have immunosuppressive and platelet aggregative capabilities. The fatty acid composition of adipose tissue reflects the dietary consumption of essential fatty acids over a period of years. Biochemical techniques have been used in epidemiological studies to accurately estimate fatty acid consumption. However, analytical epidemiology studies that have used biochemical measurements of adipose tissue fatty acid composition, have not supported a relationship between consumption of these essential fatty acids and breast cancer risk. PMID:7612909

  3. The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

    PubMed

    Ferguson, S J; Lloyd, W J; Lyons, M H; Radda, G K

    1975-05-01

    1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents. PMID:238839

  4. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  5. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  6. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  7. Child Stunting is Associated with Low Circulating Essential Amino Acids

    PubMed Central

    Semba, Richard D.; Shardell, Michelle; Sakr Ashour, Fayrouz A.; Moaddel, Ruin; Trehan, Indi; Maleta, Kenneth M.; Ordiz, M. Isabel; Kraemer, Klaus; Khadeer, Mohammed A.; Ferrucci, Luigi; Manary, Mark J.

    2016-01-01

    Background Stunting affects about one-quarter of children under five worldwide. The pathogenesis of stunting is poorly understood. Nutritional interventions have had only modest effects in reducing stunting. We hypothesized that insufficiency in essential amino acids may be limiting the linear growth of children. Methods We used a targeted metabolomics approach to measure serum amino acids, glycerophospholipids, sphingolipids, and other metabolites using liquid chromatography-tandem mass spectrometry in 313 children, aged 12–59 months, from rural Malawi. Children underwent anthropometry. Findings Sixty-two percent of the children were stunted. Children with stunting had lower serum concentrations of all nine essential amino acids (tryptophan, isoleucine, leucine, valine, methionine, threonine, histidine, phenylalanine, lysine) compared with nonstunted children (p < 0.01). In addition, stunted children had significantly lower serum concentrations of conditionally essential amino acids (arginine, glycine, glutamine), non-essential amino acids (asparagine, glutamate, serine), and six different sphingolipids compared with nonstunted children. Stunting was also associated with alterations in serum glycerophospholipid concentrations. Interpretation Our findings support the idea that children with a high risk of stunting may not be receiving an adequate dietary intake of essential amino acids and choline, an essential nutrient for the synthesis of sphingolipids and glycerophospholipids. PMID:27211567

  8. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides.

    PubMed

    Mitsuhashi, Satoshi

    2014-04-01

    Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine). PMID:24679256

  9. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, W.O.

    1987-02-27

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and thence quenching the reactivity of the nitric acid prior to nitration of the mercury metal. 1 fig.

  10. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, Wilbur O.

    1989-01-01

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and then quenching the reactivity of the nitric acid prior to nitration of the mercury metal.

  11. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, Wilbur O.

    1989-12-05

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and then quenching the reactivity of the nitric acid prior to nitration of the mercury metal.

  12. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues.

    PubMed

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints. Graphical Abstract ᅟ. PMID:27324649

  13. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues

    NASA Astrophysics Data System (ADS)

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl- N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints.

  14. Essential residues in angiotensin converting enzyme: Modification with 1-fluoro-2,4-dinitrobenzene

    SciTech Connect

    Bunning, P.; Kleemann, S.G.; Riordan, J.F. )

    1990-11-01

    The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-({sup 3}H)Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modifed enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to >95{percent}) and the rest at N{epsilon}-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.

  15. Essential fatty acid nutrition of the American alligator (Alligator mississippiensis).

    PubMed

    Staton, M A; Edwards, H M; Brisbin, I L; Joanen, T; McNease, L

    1990-07-01

    The essential fatty acid (EFA) nutrition of young American alligators (Alligator mississippiensis) was examined by feeding a variety of fats/oils with potential EFA activity. Over a 12-wk period, alligators fed diets containing 2.5 or 5.0% chicken liver oil grew longer and heavier and converted feed to body mass more efficiently than alligators fed other fat/oil combinations that lacked or contained only trace amounts of arachidonic acid [20:4(n-6)]. Alligators fed an EFA-deficient diet (containing only coconut fat as the dietary fat) were the slowest-growing animals and converted feed to body mass least efficiently. However, over a 41-wk feeding period, alligators fed this diet showed no obvious external signs of deficiency other than being reduced in size and unthrifty. Fatty acid composition of heart, liver, muscle, skin and adipose tissue lipids was influenced markedly by dietary fat composition. Tissues varied significantly in response to dietary fat composition. Heart lipids contained the lowest levels of short- and medium-chain fatty acids and the highest levels of arachidonic acid. Arachidonic acid levels were less influenced by diet than were levels of other 20- and 22-carbon polyunsaturated fatty acids. Radiotracer studies indicated that linoleic acid was converted to arachidonic acid in the liver. Nevertheless, tissue arachidonic acid levels also appeared to be maintained by concentration from dietary sources and selective conservation. It appears that a dietary source of arachidonic acid may be required for a maximum rate of growth. PMID:2114472

  16. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  17. Dietary essentiality of "nutritionally non-essential amino acids" for animals and humans.

    PubMed

    Hou, Yongqing; Yin, Yulong; Wu, Guoyao

    2015-08-01

    Based on growth or nitrogen balance, amino acids (AA) had traditionally been classified as nutritionally essential (indispensable) or non-essential (dispensable) for animals and humans. Nutritionally essential AA (EAA) are defined as either those AA whose carbon skeletons cannot be synthesized de novo in animal cells or those that normally are insufficiently synthesized de novo by the animal organism relative to its needs for maintenance, growth, development, and health and which must be provided in the diet to meet requirements. In contrast, nutritionally non-essential AA (NEAA) are those AA which can be synthesized de novo in adequate amounts by the animal organism to meet requirements for maintenance, growth, development, and health and, therefore, need not be provided in the diet. Although EAA and NEAA had been described for over a century, there are no compelling data to substantiate the assumption that NEAA are synthesized sufficiently in animals and humans to meet the needs for maximal growth and optimal health. NEAA play important roles in regulating gene expression, cell signaling pathways, digestion and absorption of dietary nutrients, DNA and protein synthesis, proteolysis, metabolism of glucose and lipids, endocrine status, men and women fertility, acid-base balance, antioxidative responses, detoxification of xenobiotics and endogenous metabolites, neurotransmission, and immunity. Emerging evidence indicates dietary essentiality of "nutritionally non-essential amino acids" for animals and humans to achieve their full genetic potential for growth, development, reproduction, lactation, and resistance to metabolic and infectious diseases. This concept represents a new paradigm shift in protein nutrition to guide the feeding of mammals (including livestock), poultry, and fish. PMID:26041391

  18. Microscopic residues of bone from dissolving human remains in acids.

    PubMed

    Vermeij, Erwin; Zoon, Peter; van Wijk, Mayonne; Gerretsen, Reza

    2015-05-01

    Dissolving bodies is a current method of disposing of human remains and has been practiced throughout the years. During the last decade in the Netherlands, two cases have emerged in which human remains were treated with acid. In the first case, the remains of a cremated body were treated with hydrofluoric acid. In the second case, two complete bodies were dissolved in a mixture of hydrochloric and sulfuric acid. In both cases, a great variety of evidence was collected at the scene of crime, part of which was embedded in resin, polished, and investigated using SEM/EDX. Apart from macroscopic findings like residual bone and artificial teeth, in both cases, distinct microscopic residues of bone were found as follows: (partly) digested bone, thin-walled structures, and recrystallized calcium phosphate. Although some may believe it is possible to dissolve a body in acid completely, at least some of these microscopic residues will always be found. PMID:25677640

  19. Essential amino acids interacting with flavonoids: A theoretical approach

    NASA Astrophysics Data System (ADS)

    Codorniu-Hernández, Edelsys; Mesa-Ibirico, Ariel; Hernández-Santiesteban, Richel; Montero-Cabrera, Luis A.; Martínez-Luzardo, Francisco; Santana-Romero, Jorge L.; Borrmann, Tobias; Stohrer, Wolf-D.

    The interaction of two flavonoid species (resorcinolic and fluoroglucinolic) with the 20 essential amino acids was studied by the multiple minima hypersurface (MMH) procedures, through the AM1 and PM3 semiempirical methods. Remarkable thermodynamic data related to the properties of the molecular association of these compounds were obtained, which will be of great utility for future investigations concerning the interaction of flavonoids with proteins. These results are compared with experimental and classical force field results reported in the available literature, and new evidences and criteria are shown. The hydrophilic amino acids demonstrated high affinity in the interaction with flavonoid molecules; the complexes with lysine are especially extremely stable. An affinity order for the interaction of both flavonoid species with the essential amino acids is suggested. Our theoretical results are compared with experimental evidence on flavonoid interactions with proteins of biomedical interest.

  20. A Single Valine Residue Plays an Essential Role in Peripherin/rds Targeting to Photoreceptor Outer Segments

    PubMed Central

    Salinas, Raquel Y.; Baker, Sheila A.; Gospe, Sidney M.; Arshavsky, Vadim Y.

    2013-01-01

    Peripherin/retinal degeneration slow (rds) is an integral membrane protein specifically localized to the light-sensing organelle of the photoreceptor cell, the outer segment. Within the outer segment, peripherin is found at the edges of photoreceptor discs, where it plays a critical role in disc morphogenesis and maintenance. Peripherin loss or mutations are often associated with severe forms of visual impairments. Like all other resident outer segment proteins, peripherin is synthesized in the photoreceptor cell body and subsequently transported to the outer segment. In an effort to further examine peripherin’s delivery to outer segments, we undertook a careful examination of its targeting sequence. Using a fluorescently labeled reporter expressed in the rods of transgenic tadpoles, we narrowed peripherin’s targeting sequence to ten amino acids within its C-terminal tail. This small stretch of amino acid residues is both necessary and sufficient for outer segment targeting. We also conducted alanine scanning of all residues within this sequence and found that only a single residue, valine at position 332, is essential for outer segment targeting. This valine is conserved in all species and its mutation is sufficient to completely abrogate the targeting of full-length peripherin in mouse rods. PMID:23342122

  1. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Arsanilic acid ; tolerances for residues. 180.550 Section 180.550 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.550 Arsanilic acid ; tolerances...

  2. Electron microscopy of carbonaceous matter in Allende acid residues

    NASA Technical Reports Server (NTRS)

    Lumpkin, G. R.

    1982-01-01

    On the basis of characteristic diffuse ring diffraction patterns, much of the carbonaceous matter in a large suite of Allende acid residues has been identified as a variety of turbostratic carbon. Crystallites of this phase contain randomly stacked sp(2) hybridized carbon layers and diffraction patterns resemble those from carbon black and glassy carbon. Carbynes are probably absent, and are certainly restricted to less than 0.5% of these acid residues. The work of Ott et al. (1981) provides a basis for the possibility that turbostratic carbon is a carrier of noble gases, but an additional component - amorphous carbon - may be necessary to explain the high release temperatures of noble gases as well as the glassy character of many of the carbonaceous particles. Carbynes are considered to be questionable as important carriers of noble gases in the Allende acid residues.

  3. Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase.

    PubMed

    Ko, Jin-A; Nam, Seung-Hee; Kim, Doman; Lee, Jun-Ho; Kim, Young-Min

    2016-05-28

    A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity. PMID:26907761

  4. Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

    PubMed

    Arai, Naoki; Nishimura, Emi; Kikuchi, Yo; Ohyama, Takashi

    2015-09-11

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation. PMID:26235877

  5. Mutational analysis of the human immunodeficiency virus type 1 Rev transactivator: essential residues near the amino terminus.

    PubMed Central

    Hope, T J; McDonald, D; Huang, X J; Low, J; Parslow, T G

    1990-01-01

    The expression of certain mRNAs from human immunodeficiency virus type 1 (HIV-1) is controlled by the viral transactivator Rev, a nucleolar protein that binds a cis-acting element in these mRNAs. Rev is encoded by two viral exons that specify amino acids 1 to 26 and 27 to 116, respectively. Earlier studies have mapped essential regions of the protein that are encoded in the second exon. By further mutational analysis of Rev, we have now identified a novel locus encoded by the first exon that also is essential for transactivation in vivo. Defined by mutations at residues 14 to 20, this locus coincides with a cluster of positively charged and nonpolar amino acids that is conserved in Rev proteins of all known primate immunodeficiency viruses. Rev proteins that contained mutations at this site were defective in both nuclear localization and transactivation and did not function as trans-dominant inhibitors of wild-type Rev. Fusion of these mutants to a heterologous nuclear protein complemented the defect in localization but did not restore biological activity. Our findings suggest that this N-terminal locus may play a direct role in transactivation, perhaps contributing to essential protein-protein interactions or forming part of the RNA-binding domain of Rev. Images PMID:2120472

  6. Residual Activity of Thermally Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

    PubMed Central

    Barnhart, Benjamin J.

    1965-01-01

    Barnhart, Benjamin J. (Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.). Residual activity of thermally denatured transforming deoxyribonucleic acid from Haemophilus influenzae. J. Bacteriol. 89:1271–1279. 1965.—The level of residual transforming activity of heated deoxyribonucleic acid (DNA) (i.e., 1 to a few per cent of native DNA-transforming activity) was found to be independent of the heating and quenching temperatures and less susceptible than native or renatured DNA to heat inactivation upon prolonged heating above or below the critical melting temperature. Similar dose-response curves were obtained for inactivation by formamide of native and renatured DNA, but the residual-active material was much more resistant. Heating DNA above the Tm in the presence of 1% formaldehyde resulted in a level of residual activity 4 logs lower than that obtained without formaldehyde. Residual-active material was not inactivated by Escherichia coli phosphodiesterase, but it was susceptible to snake venom phosphodiesterase. A new genetic marker was induced in heated-quenched DNA but not in purified residual-active material following nitrous acid treatment. Residual activity was found to be less susceptible to ultraviolet inactivation and to band at a higher density region in CsCl than native DNA. In conclusion, it is suggested that the residual-active material is a structure formed by intrastrand hydrogen bonding of the separated units of heated-quenched DNA. Such a configuration would result in at least a partially double-stranded structure, which is probably the essential characteristic of the residual-active material endowing it with biological activity. PMID:14292997

  7. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  8. Lactic Acid and Biosurfactants Production from Residual Cellulose Films.

    PubMed

    Portilla Rivera, Oscar Manuel; Arzate Martínez, Guillermo; Jarquín Enríquez, Lorenzo; Vázquez Landaverde, Pedro Alberto; Domínguez González, José Manuel

    2015-11-01

    The increasing amounts of residual cellulose films generated as wastes all over the world represent a big scale problem for the meat industry regarding to environmental and economic issues. The use of residual cellulose films as a feedstock of glucose-containing solutions by acid hydrolysis and further fermentation into lactic acid and biosurfactants was evaluated as a method to diminish and revalorize these wastes. Under a treatment consisting in sulfuric acid 6% (v/v); reaction time 2 h; solid liquid ratio 9 g of film/100 mL of acid solution, and temperature 130 °C, 35 g/L of glucose and 49% of solubilized film was obtained. From five lactic acid strains, Lactobacillus plantarum was the most suitable for metabolizing the glucose generated. The process was scaled up under optimized conditions in a 2-L bioreactor, producing 3.4 g/L of biomass, 18 g/L of lactic acid, and 15 units of surface tension reduction of a buffer phosphate solution. Around 50% of the cellulose was degraded by the treatment applied, and the liqueurs generated were useful for an efficient production of lactic acid and biosurfactants using L. plantarum. Lactobacillus bacteria can efficiently utilize glucose from cellulose films hydrolysis without the need of clarification of the liqueurs. PMID:26293409

  9. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  10. Polylactic Acid-Lemongrass Essential Oil Nanocapsules with Antimicrobial Properties.

    PubMed

    Liakos, Ioannis L; Grumezescu, Alexandru Mihai; Holban, Alina Maria; Florin, Iordache; D'Autilia, Francesca; Carzino, Riccardo; Bianchini, Paolo; Athanassiou, Athanassia

    2016-01-01

    Polylactic acid was combined with lemongrass essential oil (EO) to produce functional nanocapsules (NCs). The obtained polylactic acid nanoparticles showed antimicrobial activity both with and without the presence of lemongrass oil; however, the presence of EO improved the activity of the NCs. The presence of lemongrass assisted the formation of well-separated NCs and also provided enhanced antimicrobial properties, since lemongrass is known for its antimicrobial character. Fluorescence microscopy was used to optically observe the nanoparticles and NCs and revealed the attachment of lemongrass oil with the polylactic acid NCs. Dynamic light scattering was used to determine their size. UV absorption was used to determine the exact amount of lemongrass oil found in the polylactic acid-lemongrass oil NCs, which was important for understanding the minimum inhibitory concentration for the antimicrobial experiments. A series of clinically important microbial species were used in the study and the obtained NCs proved to have very good antimicrobial properties against all tested strains. Such NCs can be used for the design of ecological strategies, based on natural alternatives, which may be efficient against severe infections, including those that involve resistant pathogens and biofilms or those with difficult to reach localization. PMID:27399724

  11. Four residues of propeptide are essential for precursor folding of nattokinase.

    PubMed

    Jia, Yan; Cao, Xinhua; Deng, Yu; Bao, Wei; Tang, Changyan; Ding, Hanjing; Zheng, Zhongliang; Zou, Guolin

    2014-11-01

    Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses. PMID:25267722

  12. The Essentiality of Arachidonic Acid in Infant Development

    PubMed Central

    Hadley, Kevin B.; Ryan, Alan S.; Forsyth, Stewart; Gautier, Sheila; Salem, Norman

    2016-01-01

    Arachidonic acid (ARA, 20:4n-6) is an n-6 polyunsaturated 20-carbon fatty acid formed by the biosynthesis from linoleic acid (LA, 18:2n-6). This review considers the essential role that ARA plays in infant development. ARA is always present in human milk at a relatively fixed level and is accumulated in tissues throughout the body where it serves several important functions. Without the provision of preformed ARA in human milk or infant formula the growing infant cannot maintain ARA levels from synthetic pathways alone that are sufficient to meet metabolic demand. During late infancy and early childhood the amount of dietary ARA provided by solid foods is low. ARA serves as a precursor to leukotrienes, prostaglandins, and thromboxanes, collectively known as eicosanoids which are important for immunity and immune response. There is strong evidence based on animal and human studies that ARA is critical for infant growth, brain development, and health. These studies also demonstrate the importance of balancing the amounts of ARA and DHA as too much DHA may suppress the benefits provided by ARA. Both ARA and DHA have been added to infant formulas and follow-on formulas for more than two decades. The amounts and ratios of ARA and DHA needed in infant formula are discussed based on an in depth review of the available scientific evidence. PMID:27077882

  13. The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen*

    PubMed Central

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-01-01

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  14. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

    PubMed

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-09-12

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  15. Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.

    PubMed Central

    Rasche, M E; Smith, K S; Ferry, J G

    1997-01-01

    Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA. PMID:9401029

  16. Methodology for detecting residual phosphoric acid in polybenzoxazole fibers.

    PubMed

    Park, Eun Su; Sieber, John; Guttman, Charles; Rice, Kirk; Flynn, Kathleen; Watson, Stephanie; Holmes, Gale

    2009-12-01

    Because of the premature failure of in-service soft-body armor containing the ballistic fiber poly[(benzo-[1,2-d:5,4-d']-benzoxazole-2,6-diyl)-1,4-phenylene] (PBO), the Office of Law Enforcement Standards (OLES) at the National Institute of Standards and Technology (NIST) initiated a research program to investigate the reasons for this failure and to develop testing methodologies and protocols to ensure that these types of failures do not reoccur. In a report that focused on the stability of the benzoxazole ring that is characteristic of PBO fibers, Holmes, G. A.; Rice, K.; Snyder, C. R. J. Mater. Sci. 2006, 41, 4105-4116, showed that the benzoxazole ring was susceptible to hydrolytic degradation under acid conditions. Because of the processing conditions for the fibers, it is suspected by many researchers that residual phosphoric acid may cause degradation of the benzoxazole ring resulting in a reduction of ballistic performance. Prior to this work, no definitive data have indicated the presence of phosphoric acid since the residual phosphorus is not easily extracted and the processed fibers are known to incorporate phosphorus containing processing aids. Methods to efficiently extract phosphorus from PBO are described in this article. Further, characterization determined that the majority of the extractable phosphorus in PBO was attributed to the octyldecyl phosphate processing aid with some phosphoric acid being detected. Analysis by matrix assisted laser desorption ionization of model PBO oligomers indicates that the nonextractable phosphorus is attached to the PBO polymer chain as a monoaryl phosphate ester. The response of model aryl phosphates to NaOH exposure indicates that monoaryl phosphate ester is stable to NaOH washes used in the manufacturing process to neutralize the phosphoric acid reaction medium and to extract residual phosphorus impurities. PMID:19899783

  17. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.

    PubMed

    Doerfel, Lili K; Wohlgemuth, Ingo; Kothe, Christina; Peske, Frank; Urlaub, Henning; Rodnina, Marina V

    2013-01-01

    Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells. PMID:23239624

  18. XPS and STEM studies of Allende acid insoluble residues

    NASA Technical Reports Server (NTRS)

    Housley, R. M.; Clarke, D. R.

    1980-01-01

    Data on Allende acid residues obtained both before and after etching with hot HNO3 are presented. X-ray photoelectron spectra show predominantly carbonaceous material plus Fe-deficient chromite in both cases. The HNO3 oxidizes the carbonaceous material to some extent. The small chromites in these residues have a wide range of compositions somewhat paralleling those observed in larger Allende chromites and in Murchison chromites, especially in the high Al contents; however, they are deficient in divalent cations, which makes them metastable and indicates that they must have formed at relatively low temperatures. It is suggested that they formed by precipitation of Cr(3+) and Fe(3+) from olivine at low temperature or during rapid cooling.

  19. Amino acid residue doublet propensity in the protein-RNA interface and its application to RNA interface prediction.

    PubMed

    Kim, Oanh T P; Yura, Kei; Go, Nobuhiro

    2006-01-01

    Protein-RNA interactions play essential roles in a number of regulatory mechanisms for gene expression such as RNA splicing, transport, translation and post-transcriptional control. As the number of available protein-RNA complex 3D structures has increased, it is now possible to statistically examine protein-RNA interactions based on 3D structures. We performed computational analyses of 86 representative protein-RNA complexes retrieved from the Protein Data Bank. Interface residue propensity, a measure of the relative importance of different amino acid residues in the RNA interface, was calculated for each amino acid residue type (residue singlet interface propensity). In addition to the residue singlet propensity, we introduce a new residue-based propensity, which gives a measure of residue pairing preferences in the RNA interface of a protein (residue doublet interface propensity). The residue doublet interface propensity contains much more information than the sum of two singlet propensities alone. The prediction of the RNA interface using the two types of propensities plus a position-specific multiple sequence profile can achieve a specificity of about 80%. The prediction method was then applied to the 3D structure of two mRNA export factors, TAP (Mex67) and UAP56 (Sub2). The prediction enables us to point out candidate RNA interfaces, part of which are consistent with previous experimental studies and may contribute to elucidation of atomic mechanisms of mRNA export. PMID:17130160

  20. Chemical and isotopic compositions in acid residues from various meteorites

    NASA Technical Reports Server (NTRS)

    Kano, N.; Yamakoshi, K.; Matsuzaki, H.; Nogami, K.

    1993-01-01

    We are planning to carry out systematic isotopic investigations of Ru, Mg, etc., in primordial samples. The investigations will be pursued in the context of a study of the pre-history of the solar system. It is hoped that the study will yield direct evidence for processes of nucleosynthesis in the pre-solar stage and detection of extinct radioactive nuclides. In this paper, we present the results of chemical compositions of acid residues obtained from three types of meteorites: Canyon Diablo (IA), Allende (CV3), and Nuevo Mercuro (H5); and the preliminary results of Ru isotopic compositions.

  1. Essential fatty acid deficiency in mice impairs lactose digestion.

    PubMed

    Lukovac, S; Los, E L; Stellaard, F; Rings, E H H M; Verkade, H J

    2008-09-01

    Essential fatty acid (EFA) deficiency in mice induces fat malabsorption. We previously reported indications that the underlying mechanism is located at the level of the intestinal mucosa. We have investigated the effects of EFA deficiency on small intestinal morphology and function. Mice were fed an EFA-deficient or control diet for 8 wk. A 72-h fat balance, the EFA status, and small intestinal histology were determined. Carbohydrate absorptive and digestive capacities were assessed by stable isotope methodology after administration of [U-(13)C]glucose and [1-(13)C]lactose. The mRNA expression and enzyme activity of lactase, and concentrations of the EFA linoleic acid (LA) were measured in small intestinal mucosa. Mice fed the EFA-deficient diet were markedly EFA-deficient with a profound fat malabsorption. EFA deficiency did not affect the histology or proliferative capacity of the small intestine. Blood [13C6]glucose appearance and disappearance were similar in both groups, indicating unaffected monosaccharide absorption. In contrast, blood appearance of [13C]glucose, originating from [1-(13)C]lactose, was delayed in EFA-deficient mice. EFA deficiency profoundly reduced lactase activity (-58%, P<0.01) and mRNA expression (-55%, P<0.01) in mid-small intestine. Both lactase activity and its mRNA expression strongly correlated with mucosal LA concentrations (r=0.77 and 0.79, respectively, P<0.01). EFA deficiency in mice inhibits the capacity to digest lactose but does not affect small intestinal histology. These data underscore the observation that EFA deficiency functionally impairs the small intestine, which in part may be mediated by low LA levels in the enterocytes. PMID:18653724

  2. Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus.

    PubMed

    Sands, B; Ellse, L; Wall, R

    2016-03-01

    Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment. PMID:26522385

  3. Oxolinic acid in the trout: bioavailability and tissue residues.

    PubMed

    Archimbault, P; Ambroggi, G; Nicolas, S

    1988-01-01

    A study was performed on the serum bioavailability and tissue elimination of oxolinic acid in the trout. The compound was added to the diet and administered at a dosage-level of 12 mg/kg/day for 7 consecutive days. The study utilized an analytical technique, high performance liquid chromatography, which has been described in detail here. The results obtained demonstrate that serum concentrations higher than the MIC for the control of the target pathogens (Aeromonas and Yersinia) can by sustained throughout the treatment period. The same positive results were observed in the tissues. Besides, on the base of a tolerance level of 0.05 ppm for the residue levels of oxolinic acid in the edible tissues (muscle mass and skin), a withdrawal time of six days after interruption of the prescribed treatment can be proposed. PMID:3400972

  4. Essential amino acids: master regulators of nutrition and environmental footprint?

    PubMed

    Tessari, Paolo; Lante, Anna; Mosca, Giuliano

    2016-01-01

    The environmental footprint of animal food production is considered several-fold greater than that of crops cultivation. Therefore, the choice between animal and vegetarian diets may have a relevant environmental impact. In such comparisons however, an often neglected issue is the nutritional value of foods. Previous estimates of nutrients' environmental footprint had predominantly been based on either food raw weight or caloric content, not in respect to human requirements. Essential amino acids (EAAs) are key parameters in food quality assessment. We re-evaluated here the environmental footprint (expressed both as land use for production and as Green House Gas Emission (GHGE), of some animal and vegetal foods, titrated to provide EAAs amounts in respect to human requirements. Production of high-quality animal proteins, in amounts sufficient to match the Recommended Daily Allowances of all the EAAs, would require a land use and a GHGE approximately equal, greater o smaller (by only ±1-fold), than that necessary to produce vegetal proteins, except for soybeans, that exhibited the smallest footprint. This new analysis downsizes the common concept of a large advantage, in respect to environmental footprint, of crops vs. animal foods production, when human requirements of EAAs are used for reference. PMID:27221394

  5. Essential amino acids: master regulators of nutrition and environmental footprint?

    PubMed Central

    Tessari, Paolo; Lante, Anna; Mosca, Giuliano

    2016-01-01

    The environmental footprint of animal food production is considered several-fold greater than that of crops cultivation. Therefore, the choice between animal and vegetarian diets may have a relevant environmental impact. In such comparisons however, an often neglected issue is the nutritional value of foods. Previous estimates of nutrients’ environmental footprint had predominantly been based on either food raw weight or caloric content, not in respect to human requirements. Essential amino acids (EAAs) are key parameters in food quality assessment. We re-evaluated here the environmental footprint (expressed both as land use for production and as Green House Gas Emission (GHGE), of some animal and vegetal foods, titrated to provide EAAs amounts in respect to human requirements. Production of high-quality animal proteins, in amounts sufficient to match the Recommended Daily Allowances of all the EAAs, would require a land use and a GHGE approximately equal, greater o smaller (by only ±1-fold), than that necessary to produce vegetal proteins, except for soybeans, that exhibited the smallest footprint. This new analysis downsizes the common concept of a large advantage, in respect to environmental footprint, of crops vs. animal foods production, when human requirements of EAAs are used for reference. PMID:27221394

  6. The role of essential fatty acids in development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty acids are aliphatic monocarboxylic acids. They are classified as saturated, monounsaturated, or polyunsaturated fatty acids depending upon the number of double bonds in the carbon chain. Saturated fatty acids have no double bonds, monounsaturated fatty acids have 1 double bond, and polyunsat...

  7. A Soluble, Folded Protein without Charged Amino Acid Residues.

    PubMed

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall; Johansson, Kristoffer Enøe; Villa, Mara; Willemoës, Martin; Lindorff-Larsen, Kresten; Teilum, Kaare; Winther, Jakob R

    2016-07-19

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced. PMID:27307139

  8. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  9. Removal of coagulant aluminum from water treatment residuals by acid.

    PubMed

    Okuda, Tetsuji; Nishijima, Wataru; Sugimoto, Mayo; Saka, Naoyuki; Nakai, Satoshi; Tanabe, Kazuyasu; Ito, Junki; Takenaka, Kenji; Okada, Mitsumasa

    2014-09-01

    Sediment sludge during coagulation and sedimentation in drinking water treatment is called "water treatment residuals (WTR)". Polyaluminum chloride (PAC) is mainly used as a coagulant in Japan. The recycling of WTR has been desired; one method for its reuse is as plowed soil. However, WTR reuse in this way is inhibited by the aluminum from the added PAC, because of its high adsorption capacity for phosphate and other fertilizer components. The removal of such aluminum from WTR would therefore be advantageous for its reuse as plowed soil; this research clarified the effect of acid washing on aluminum removal from WTR and on plant growth in the treated soil. The percentage of aluminum removal from raw WTR by sulphuric acid solution was around 90% at pH 3, the percentage decreasing to 40% in the case of a sun-dried sample. The maximum phosphate adsorption capacity was decreased and the available phosphorus was increased by acid washing, with 90% of aluminum removal. The enhancement of Japanese mustard spinach growth and the increased in plant uptake of phosphates following acid washing were observed. PMID:24835954

  10. Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

    PubMed Central

    Miller, Janet C.; Waley, S. G.

    1971-01-01

    1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes. PMID:5165707

  11. Octanoic acid in alcohol-responsive essential tremor

    PubMed Central

    McCrossin, Gayle; Lungu, Codrin; Considine, Elaine; Toro, Camilo; Nahab, Fatta B.; Auh, Sungyoung; Buchwald, Peter; Grimes, George J.; Starling, Judith; Potti, Gopal; Scheider, Linda; Kalowitz, Daniel; Bowen, Daniel; Carnie, Andrea; Hallett, Mark

    2013-01-01

    Objective: To assess safety and efficacy of an oral, single, low dose of octanoic acid (OA) in subjects with alcohol-responsive essential tremor (ET). Methods: We conducted a double-blind, placebo-controlled, crossover, phase I/II clinical trial evaluating the effect of 4 mg/kg OA in 19 subjects with ET. The primary outcome was accelerometric postural tremor power of the dominant hand 80 minutes after administration. Secondary outcomes included digital spiral analysis, pharmacokinetic sampling, as well as safety measures. Results: OA was safe and well tolerated. Nonserious adverse events were mild (Common Terminology Criteria for Adverse Events grade 1) and equally present after OA and placebo. At the primary outcome, OA effects were not different from placebo. Secondary outcome analyses of digital spiral analysis, comparison across the entire time course in weighted and nonweighted accelerometry, as well as nondominant hand tremor power did not show a benefit of OA over placebo. The analysis of individual time points showed that OA improved tremor at 300 minutes (dominant hand, F1,16 = 5.49, p = 0.032 vs placebo), with a maximum benefit at 180 minutes after OA (both hands, F1,16 = 6.1, p = 0.025). Conclusions: Although the effects of OA and placebo at the primary outcome were not different, secondary outcome measures suggest superiority of OA in reducing tremor at later time points, warranting further trials at higher dose levels. Classification of evidence: This study provides Class I evidence that a single 4-mg/kg dose of OA is not effective in reducing postural tremor in patients with ET at a primary outcome of 80 minutes, but is effective for a secondary outcome after 180 minutes. PMID:23408867

  12. Phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.

    PubMed

    Pailler, Jérémy; Aucher, Willy; Pires, Magali; Buddelmeijer, Nienke

    2012-05-01

    Lgt of Escherichia coli catalyzes the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth, as demonstrated here by the analysis of an lgt depletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein. Its membrane topology was determined by fusing Lgt to β-galactosidase and alkaline phosphatase and by substituted cysteine accessibility method (SCAM) studies. The data show that Lgt is embedded in the membrane by seven transmembrane segments, that its N terminus faces the periplasm, and that its C terminus faces the cytoplasm. Highly conserved amino acids in Lgt of both Gram-negative and Gram-positive bacteria were identified. Lgt enzymes are characterized by a so-called Lgt signature motif in which four residues are invariant. Ten conserved residues were replaced with alanine, and the activity of these Lgt variants was analyzed by their ability to complement the lgt depletion strain. Residues Y26, N146, and G154 are absolutely required for Lgt function, and R143, E151, R239, and E243 are important. The results demonstrate that the majority of the essential residues of Lgt are located in the membrane and that the Lgt signature motif faces the periplasm. PMID:22287519

  13. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  14. The Nature of Intermolecular Interactions Between Aromatic Amino Acid Residues

    SciTech Connect

    Gervasio, Francesco; Chelli, Riccardo; Procacci, Piero; Schettino, Vincenzo

    2002-05-01

    The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single-point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic-aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures.

  15. 40 CFR 180.325 - 2-(m-Chlorophenoxy) propionic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 2-(m-Chlorophenoxy) propionic acid... Tolerances § 180.325 2-(m-Chlorophenoxy) propionic acid; tolerances for residues. (a) General. A tolerance is established for negligible residues of the plant regulator 2-(m-chlorophenoxy) propionic acid from...

  16. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  17. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  18. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  19. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  20. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  1. Lower fetal status of docosahexaenoic acid, arachidonic acid and essential fatty acids is associated with less favorable neonatal neurological condition.

    PubMed

    Dijck-Brouwer, D A Janneke; Hadders-Algra, Mijna; Bouwstra, Hylco; Decsi, Tamás; Boehm, Günther; Martini, Ingrid A; Boersma, E Rudy; Muskiet, Frits A J

    2005-01-01

    Long-chain polyunsaturated fatty acids, notably arachidonic (AA) and docosahexaenoic (DHA) acids are abundant in brain and may be conditionally essential in fetal life. We investigated umbilical artery (UA) and vein (UV) fatty acid compositions and early neonatal neurological condition in 317 term infants. Neurological condition was summarized as a clinical classification and a 'neurological optimality score' (NOS). Neurologically abnormal infants (n=27) had lower UV DHA and essential fatty acid (EFA) status. NOS correlated positively with AA (UV), and EFA (UV) and DHA status (UV and UA) and negatively with 18:2omega6 and omega9 (UV), and 20:3omega9, omega7 and C18 trans fatty acids (UV and UA). UV DHA, AA, saturated fatty acids, gestational age and obstetrical optimality score explained 16.2% of the NOS variance. Early postnatal neurological condition seems negatively influenced by lower fetal DHA, AA and EFA status. C18 trans fatty acids and 18:2omega6 may exert negative effects by impairment of LCP status. PMID:15589396

  2. The Arginine Residue within the C-Terminal Active Core of Bombyx mori Pheromone Biosynthesis-Activating Neuropeptide is Essential for Receptor Binding and Activation

    PubMed Central

    Kawai, Takeshi; Lee, Jae Min; Nagata, Koji; Matsumoto, Shogo; Tanokura, Masaru; Nagasawa, Hiromichi

    2012-01-01

    In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). Bombyx mori PBAN (BomPBAN) consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R) residue at the second position from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to clarify the role of the Arg residue in the BomPBAN active core. We synthesized 10-residue peptides corresponding to the C-terminal part of BomPBAN with a series of replacements at the second position from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells expressing a fluorescent PBAN receptor chimera (PBANR–EGFP) using the fluorescent Ca2+ indicator, Fura Red–AM. The PBAN analogs with the C2 position replaced with alanine (Ala, A), aspartic acid (Asp, D), serine (Ser, S), or l-2-aminooctanoic acid (Aoc) decreased PBAN-like activity. RC2A (SKTRYFSPALamide) and RC2D (SKTRYFSPDLamide) had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide). We also prepared Rhodamine Red-labeled peptides of the PBAN analogs and examined their ability to bind PBANR. In contrast to Rhodamine Red-PBAN C10 at 100 nM, none of the synthetic analogs exhibited PBANR binding at the same concentration. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation. PMID:22654866

  3. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.

    PubMed

    Nguyen, Long N; Ma, Dongliang; Shui, Guanghou; Wong, Peiyan; Cazenave-Gassiot, Amaury; Zhang, Xiaodong; Wenk, Markus R; Goh, Eyleen L K; Silver, David L

    2014-05-22

    Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is essential for normal brain growth and cognitive function. Consistent with its importance in the brain, DHA is highly enriched in brain phospholipids. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be de novo synthesized in brain and must be imported across the blood-brain barrier, but mechanisms for DHA uptake in brain have remained enigmatic. Here we identify a member of the major facilitator superfamily--Mfsd2a (previously an orphan transporter)--as the major transporter for DHA uptake into brain. Mfsd2a is found to be expressed exclusively in endothelium of the blood-brain barrier of micro-vessels. Lipidomic analysis indicates that Mfsd2a-deficient (Mfsd2a-knockout) mice show markedly reduced levels of DHA in brain accompanied by neuronal cell loss in hippocampus and cerebellum, as well as cognitive deficits and severe anxiety, and microcephaly. Unexpectedly, cell-based studies indicate that Mfsd2a transports DHA in the form of lysophosphatidylcholine (LPC), but not unesterified fatty acid, in a sodium-dependent manner. Notably, Mfsd2a transports common plasma LPCs carrying long-chain fatty acids such LPC oleate and LPC palmitate, but not LPCs with less than a 14-carbon acyl chain. Moreover, we determine that the phosphor-zwitterionic headgroup of LPC is critical for transport. Importantly, Mfsd2a-knockout mice have markedly reduced uptake of labelled LPC DHA, and other LPCs, from plasma into brain, demonstrating that Mfsd2a is required for brain uptake of DHA. Our findings reveal an unexpected essential physiological role of plasma-derived LPCs in brain growth and function. PMID:24828044

  4. Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii

    PubMed Central

    Lim, Jong-Min; Vikramathithan, Jayaraman; Hwangbo, Kwon; Ahn, Joon-Woo; Park, Youn-Il; Choi, Dong-Woog; Jeong, Won-Joong

    2015-01-01

    Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7. PMID:25699037

  5. Dietary essential fatty acids change the fatty acid profile of rat neural mitochondria over time.

    PubMed

    Dyer, J R; Greenwood, C E

    1991-10-01

    This experiment examined the time course over which the amount of dietary essential fatty acids (EFA) affects brain mitochondrial fatty acids. Weanling rats were fed 20% (wt/wt) fat diets that contained either 4 or 15% (wt/wt of diet) EFA for 1, 2, 3 or 6 wk or a 10% EFA diet for 3 or 6 wk. The EFA ratio [18:2(n-6)/18:3(n-3)] of all diets was approximately 30. Fatty acid analysis of brain mitochondrial phosphatidylethanolamine, phosphatidylcholine and cardiolipin revealed that the largest dietary effect was on 18:2(n-6), which was 30% higher in rats fed the 15 vs. 4% EFA diets after 1 wk. This difference increased to twofold by 3 wk and was still twofold after 6 wk. These results demonstrate several facts: 1) the response of 18:2(n-6) in cardiolipin to dietary EFA is very fast and large, relative to changes in other quantitatively major fatty acids observed in weanling rats; 2) the 18:2(n-6) level in neural cardiolipin stabilizes after 3 wk of feeding at a level dependent upon the amount of dietary EFA; and 3) at least one neural fatty acid, 18:2(n-6), is very sensitive to amounts of dietary EFA that are well above the animal's EFA requirement. PMID:1765818

  6. A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

    PubMed Central

    2015-01-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  7. The role of essential fatty acids in development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of docosahexaenoic acid (DHA) and arachidonic acid (ARA) in human milk but not in infant formula, coupled with lower plasma and brain lipid contents of DHA in formula-fed than in breast-fed infants and reports of higher IQ in individuals who were breast-fed versus formula-fed as infants...

  8. Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum.

    PubMed

    Huang, Yuanyuan; Zhang, Hao; Tian, Hongming; Li, Cheng; Han, Shuangyan; Lin, Ying; Zheng, Suiping

    2015-09-01

    N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine that is inhibited by its end product L-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized L-arginine producing strain. PMID:25750030

  9. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... established for the combined residues, free and conjugated, of the herbicide MCPB, 4-(4-chloro-2-methylphenoxy)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following...

  10. Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.

    PubMed Central

    Song, D D; Jacques, N A

    1999-01-01

    The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius. PMID:10548559

  11. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Tolerances § 180.155 1-Naphthaleneacetic acid; tolerances for residues. (a) General. Tolerances are... Avocado 0.05 12/31/12 (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  12. Pyrolytic characteristics of biomass acid hydrolysis residue rich in lignin.

    PubMed

    Huang, Yanqin; Wei, Zhiguo; Yin, Xiuli; Wu, Chuangzhi

    2012-01-01

    Pyrolytic characteristics of acid hydrolysis residue (AHR) of corncob and pinewood (CAHR, WAHR) were investigated using a thermo-gravimetric analyzer (TGA) and a self-designed pyrolysis apparatus. Gasification reactivity of CAHR char was then examined using TGA and X-ray diffractometer. Result of TGA showed that thermal degradation curves of AHR descended smoothly along with temperature increasing from 150 °C to 850 °C, while a "sharp mass loss stage" for original biomass feedstock (OBF) was observed. Char yield from AHR (42.64-30.35 wt.%) was found to be much greater than that from OBF (26.4-19.15 wt.%). In addition, gasification reactivity of CAHR char was lower than that of corncob char, and there was big difference in micro-crystallite structure. It was also found that CAHR char reactivity decreased with pyrolysis temperature, but increased with pyrolysis heating rate and gasification temperature at 850-950 °C. Furthermore, CAHR char reactivity performed better under steam atmosphere than under CO2 atmosphere. PMID:22055106

  13. Oxidation in Acidic Medium of Lignins from Agricultural Residues

    NASA Astrophysics Data System (ADS)

    Labat, Gisele Aparecida Amaral; Gonçalves, Adilson Roberto

    Agricultural residues as sugarcane straw and bagasse are burned in boilers for generation of energy in sugar and alcohol industries. However, excess of those by-products could be used to obtain products with higher value. Pulping process generates cellulosic pulps and lignin. The lignin could be oxidized and applied in effluent treatments for heavy metal removal. Oxidized lignin presents very strong chelating properties. Lignins from sugarcane straw and bagasse were obtained by ethanol-water pulping. Oxidation of lignins was carried out using acetic acid and Co/Mn/Br catalytical system at 50, 80, and 115 °C for 5 h. Kinetics of the reaction was accomplished by measuring the UV-visible region. Activation energy was calculated for lignins from sugarcane straw and bagasse (34.2 and 23.4 kJ mol-1, respectively). The first value indicates higher cross-linked formation. Fourier-transformed infrared spectroscopy data of samples collected during oxidation are very similar. Principal component analysis applied to spectra shows only slight structure modifications in lignins after oxidation reaction.

  14. Negative Confounding by Essential Fatty Acids in Methylmercury Neurotoxicity Associations

    PubMed Central

    Choi, Anna L; Mogensen, Ulla B.; Bjerve, Kristian S.; Debes, Frodi; Weihe, Pal; Grandjean, Philippe; Budtz-Jørgensen, Esben

    2014-01-01

    Background Methylmercury, a worldwide contaminant of fish and seafood, can cause adverse effects on the developing nervous system. However, long-chain n-3 polyunsaturated fatty acids in seafood provide beneficial effects on brain development. Negative confounding will likely result in underestimation of both mercury toxicity and nutrient benefits unless mutual adjustment is included in the analysis. Methods We examined these associations in 176 Faroese children, in whom prenatal methylmercury exposure was assessed from mercury concentrations in cord blood and maternal hair. The relative concentrations of fatty acids were determined in cord serum phospholipids. Neuropsychological performance in verbal, motor, attention, spatial, and memory functions was assessed at 7 years of age. Multiple regression and structural equation models (SEMs) were carried out to determine the confounder-adjusted associations with methylmercury exposure. Results A short delay recall (in percent change) in the California Verbal Learning Test (CVLT) was associated with a doubling of cord blood methylmercury (−18.9, 95% confidence interval [CI] = −36.3, −1.51). The association became stronger after the inclusion of fatty acid concentrations in the analysis (−22.0, 95% confidence interval [CI] = −39.4, −4.62). In structural equation models, poorer memory function (corresponding to a lower score in the learning trials and short delay recall in CVLT) was associated with a doubling of prenatal exposure to methylmercury after the inclusion of fatty acid concentrations in the analysis (−1.94, 95% CI = −3.39, −0.49). Conclusions Associations between prenatal exposure to methylmercury and neurobehavioral deficits in memory function at school age were strengthened after fatty acid adjustment, thus suggesting that n-3 fatty acids need to be included in analysis of similar studies to avoid underestimation of the associations with methylmercury exposure. PMID:24561639

  15. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    PubMed

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production. PMID:27474618

  16. Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation.

    PubMed

    Pekovic, Vanja; Gibbs-Seymour, Ian; Markiewicz, Ewa; Alzoghaibi, Fahad; Benham, Adam M; Edwards, Robert; Wenhert, Manfred; von Zglinicki, Thomas; Hutchison, Christopher J

    2011-12-01

    Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence. PMID:21951640

  17. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  18. Essential fatty acid intake and serum fatty acid composition among adolescent girls in central Mozambique.

    PubMed

    Freese, Riitta; Korkalo, Liisa; Vessby, Bengt; Tengblad, Siv; Vaara, Elina M; Hauta-alus, Helena; Selvester, Kerry; Mutanen, Marja

    2015-04-14

    Many African diets are low in fat but are currently changing because of nutrition transition. We studied fat and fatty acid (FA) intake and the essential fatty acid (EFA) status of adolescent girls (aged 14-19 years, n 262) in Zambezia Province, central Mozambique. A cross-sectional study was carried out in a city as well as in the towns and rural villages of a coastal and an inland district. Dietary intake and FA sources were studied in a 24 h dietary recall. FA compositions of cholesteryl esters and phospholipids of non-fasting serum samples were analysed by GLC. Fat intake was low (13-18 % of energy) in all areas. Coconut and palm oil were the main sources of fat, and soyabean oil and maize were the main sources of PUFA. Compared to Food and Agriculture Organization/WHO 2010 recommendations, intake of linoleic acid (LA, 18 : 2n-6) was inadequate in the coastal district, and intakes of n-3 PUFA were inadequate in all areas. FA compositions of serum lipids differed between areas. The proportions of LA tended to be highest in the city and lowest in the rural areas. The phospholipid mead (20 : 3n-9):arachidonic acid (20 : 4n-6) ratio did not indicate EFA insufficiency. LA proportions in phospholipids were low, but those of long-chain n-6 and n-3 PUFA were high in comparison with Western adolescents. To conclude, fat sources, FA intake and EFA status differed between adolescent girls living in different types of communities. Fat intake was low, but EFA insufficiency was not indicated. PMID:25772191

  19. Identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity.

    PubMed

    Lee, J; Gravel, M; Gao, E; O'Neill, R C; Braun, P E

    2001-05-01

    2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis. PMID:11278504

  20. Content of essential polyunsaturated fatty acids in three canned fish species.

    PubMed

    Gladyshev, Michail I; Sushchik, Nadezhda N; Makhutova, Olesia N; Kalachova, Galina S

    2009-05-01

    Three canned fish species--Pacific saury (Cololabis saira), Pacific herring (Clupea harengus) and Baltic sprat (Sprattus sprattus)--most common and popular in Russia, were analyzed for fatty acids. Special attention was paid to long-chain essential polyunsaturated fatty acids: eicosapentaenoic acid (20:5omega3) and docosahexaenoic acid (22:6omega3). Sums of eicosapentaenoic acid and docosahexaenoic acid in saury, herring and sprat were, on average, 2.42, 1.80 and 1.43 g/100 g product, respectively. Contents of these essential acids in all the canned fish species were found to be very high compared with many other fish reported in the available literature. All the canned fish appeared to be highly valuable products for human nutrition concerning the content of eicosapentaenoic and docosahexaenoic acids. PMID:18608541

  1. Amino acid residues required for fast Na(+)-channel inactivation: charge neutralizations and deletions in the III-IV linker.

    PubMed Central

    Patton, D E; West, J W; Catterall, W A; Goldin, A L

    1992-01-01

    The cytoplasmic linker connecting domains III and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain IIA Na+ channel, it consists of 53 amino acids of which 15 are charged. To investigate the role of this linker in inactivation, we mutated all 15 of the charged residues in various combinations. All but one of these mutants expressed functional channels, and all of these inactivated with kinetics similar to the wild-type channel. We then constructed a series of deletion mutations that span the III-IV linker to determine if any region of the linker is essential for fast inactivation. Deletion of the first 10 amino acids completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had no substantial effect on inactivation. These results demonstrate that some residues in the amino end of the III-IV linker are critical for fast Na(+)-channel inactivation, but that the highly conserved positively charged and paired negatively charged residues are not essential. PMID:1332059

  2. Chlorine residuals and haloacetic acid reduction in rapid sand filtration.

    PubMed

    Chuang, Yi-Hsueh; Wang, Gen-Shuch; Tung, Hsin-hsin

    2011-11-01

    It is quite rare to find biodegradation in rapid sand filtration for drinking water treatment. This might be due to frequent backwashes and low substrate levels. High chlorine concentrations may inhibit biofilm development, especially for plants with pre-chlorination. However, in tropical or subtropical regions, bioactivity on the sand surface may be quite significant due to high biofilm development--a result of year-round high temperature. The objective of this study is to explore the correlation between biodegradation and chlorine concentration in rapid sand filters, especially for the water treatment plants that practise pre-chlorination. In this study, haloacetic acid (HAA) biodegradation was found in conventional rapid sand filters practising pre-chlorination. Laboratory column studies and field investigations were conducted to explore the association between the biodegradation of HAAs and chlorine concentrations. The results showed that chlorine residual was an important factor that alters bioactivity development. A model based on filter influent and effluent chlorine was developed for determining threshold chlorine for biodegradation. From the model, a temperature independent chlorine concentration threshold (Cl(threshold)) for biodegradation was estimated at 0.46-0.5mgL(-1). The results imply that conventional filters with adequate control could be conducive to bioactivity, resulting in lower HAA concentrations. Optimizing biodegradable disinfection by-product removal in conventional rapid sand filter could be achieved with minor variation and a lower-than-Cl(threshold) influent chlorine concentration. Bacteria isolation was also carried out, successfully identifying several HAA degraders. These degraders are very commonly seen in drinking water systems and can be speculated as the main contributor of HAA loss. PMID:21974919

  3. The acid lability of the glycosidic bonds of L-iduronic acid residues in glycosaminoglycans.

    PubMed Central

    Conrad, H E

    1980-01-01

    Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers. PMID:6453583

  4. Analysis of essential oils and fatty acids from Platycodi Radix using chemometric methods and retention indices.

    PubMed

    He, Min; Li, Yaping; Yan, Jun; Cao, Dongsheng; Liang, Yizeng

    2013-04-01

    The chemical composition of the essential oils and fatty acids among nine groups of Platycodi Radix in China was analyzed by gas chromatography-mass spectrometry. Complicated components were resolved using chemometric methods. Simultaneously, the various features among heuristic evolving latent projections, selective ion analysis and alternative moving window factor analysis were compared by using some experimental data. Temperature-programmed retention indices were applied in further identification of the chemical composition of the essential oils. The equivalent chain length, fraction chain length, and an established special retention indices library integrated with mass spectrometry were also applied to further identify the composition of fatty acids, including total fatty acids, esterified fatty acids and free fatty acids. A total of 121 different compounds accounting for 95.12-98.74% were identified among the essential oils. Chemical polymorphisms and variation existed in the essential oils of Platycodi Radix. Sixteen components were identified in fatty acids, and linoleic acid (18:2n-6c) and other unsaturated acid possess a characteristic majority. PMID:22964951

  5. Essential Fatty Acids and Attention-Deficit-Hyperactivity Disorder: A Systematic Review

    ERIC Educational Resources Information Center

    Raz, Raanan; Gabis, Lidia

    2009-01-01

    Aim: Essential fatty acids (EFAs), also known as omega-3 and omega-6 fatty acids, have been claimed to have beneficial effects as a treatment for attention-deficit-hyperactivity disorder (ADHD). Animal experiments have provided information about the role of EFA in the brain, and several mechanisms of EFA activity are well known. The current review…

  6. The Regulation of Essential Amino Acid Synthesis and Accumulation in Plants.

    PubMed

    Galili, Gad; Amir, Rachel; Fernie, Alisdair R

    2016-04-29

    Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the world's major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants. PMID:26735064

  7. Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

    SciTech Connect

    Gassner, G.T.; Dickie, J.P.; Hamerski, D.A.; Magnuson, J.K.; Anderson, J.S. )

    1990-05-01

    Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. {sup 31}P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB{sup 3}H{sub 4} followed by complete acid hydrolysis yielded ({sup 3}H) glucosaminitol from the true reducing end residue of teichuronic acid and ({sup 3}H)glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

  8. The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein

    PubMed Central

    Neuberger, A.; Ratcliffe, Wendy A.

    1972-01-01

    Rabbit Tamm–Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm–Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm–Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues. PMID:4349114

  9. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

    PubMed Central

    Di Marco, A; Gloaguen, I; Graziani, R; Paonessa, G; Saggio, I; Hudson, K R; Laufer, R

    1996-01-01

    Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease. Images Fig. 1 Fig. 6 PMID:8799186

  10. Identification of essential histidine residues involved in heme binding and Hemozoin formation in heme detoxification protein from Plasmodium falciparum.

    PubMed

    Nakatani, Keisuke; Ishikawa, Haruto; Aono, Shigetoshi; Mizutani, Yasuhisa

    2014-01-01

    Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum. PMID:25138161

  11. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  12. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  13. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  14. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  15. Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

    PubMed Central

    Trujillo, Carolina; Taylor-Parker, Julian; Harrison, Robert; Murphy, John R.

    2014-01-01

    The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol. PMID:20398220

  16. Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

    PubMed

    Lu, X; Gilbert, H F; Harper, J W

    1992-05-01

    Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1567868

  17. Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues.

    PubMed

    Fischer, Klaus; Bipp, Hans-Peter

    2005-05-01

    Carbohydrate-rich biomass residues, i.e. sugar beet molasses, whey powder, wine yeast, potato peel sludge, spent hops, malt dust and apple marc, were tested as starting materials for the generation of marketable chemicals, e.g. aliphatic acids, sugar acids and mono-/disaccharides. Residues were oxidized or hydrolyzed under acidic or alkaline conditions applying conventional laboratory digestion methods and microwave assisted techniques. Yields and compositions of the oxidation products differed according to the oxidizing agent used. Main products of oxidation by 30% HNO(3) were acetic, glucaric, oxalic and glycolic acids. Applying H(2)O(2)/CuO in alkaline solution, the organic acid yields were remarkably lower with formic, acetic and threonic acids as main products. Gluconic acid was formed instead of glucaric acid throughout. Reaction of a 10% H(2)O(2) solution with sugar beet molasses generated formic and lactic acids mainly. Na(2)S(2)O(8) solutions were very inefficient at oxidizing the residues. Glucose, arabinose and galactose were formed during acidic hydrolysis of malt dust and apple marc. The glucose content reached 0.35 g per gram of residue. Important advantages of the microwave application were lower reaction times and reduced reagent demands. PMID:15607197

  18. Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol.

    PubMed

    Erkan, Naciye; Ayranci, Guler; Ayranci, Erol

    2008-09-01

    Antioxidant activities of three pure compounds: carnosic acid, rosmarinic acid and sesamol, as well as two plant extracts: rosemary extract and blackseed essential oil, were examined by applying DPPH and ABTS(+) radical-scavenging assays and the ferric thiocyanate test. All three test methods proved that rosemary extract had a higher antioxidant activity than blackseed essential oil. The order of antioxidant activity of pure compounds showed variations in different tests. This was attributed to structural factors of individual compounds. Phenolic contents of blackseed essential oil and rosemary extract were also determined. Rosemary extract was found to have a higher phenolic content than blackseed essential oil. This fact was utilised in explaining the higher antioxidant activity of rosemary extract. PMID:26050168

  19. Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine.

    PubMed

    Ugolev, Yelena; Segal, Tali; Yaffe, Dana; Gros, Yael; Schuldiner, Shimon

    2013-11-01

    Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2. PMID:24062308

  20. Essential fatty acid deficiency in patients with lesions of the gastrointestinal tract

    PubMed Central

    Wapnick, Simon; Norden, Daphne A.; Venturas, Dawn J.

    1974-01-01

    The relative amounts of unsaturated fatty acids in the serum lipids of 14 patients who have undergone small bowel resection have been investigated. These results are compared with those from control subjects and also from patients suffering from cancer of the oesophagus. Both groups of patients showed patterns of unsaturated fatty acids suggesting essential fatty acid deficiency. In the resection patients there was no correlation between the extent of intestinal removal and the extent of the change in the unsaturated fatty acid pattern. PMID:18668845

  1. Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis

    PubMed Central

    Grimek, T. L.; Holden, H.; Rayment, I.; Escalante-Semerena, J. C.

    2003-01-01

    The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ ions; equimolar concentrations of Mn2+ ions were a poor substitute for Mg2+ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The Km of PrpB for 2-MIC was measured at 19 μM, with a kcat of 105 s−1. Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpBK121A, PrpBH125A, and PrpBR122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in kcat for 2-MIC lyase activity, respectively. The PrpBD58A and PrpBC123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is

  2. Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis.

    PubMed

    Grimek, T L; Holden, H; Rayment, I; Escalante-Semerena, J C

    2003-08-01

    The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related

  3. Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.

    PubMed

    Corona, Angela; Di Leva, Francesco Saverio; Thierry, Sylvain; Pescatori, Luca; Cuzzucoli Crucitti, Giuliana; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Rigogliuso, Giuseppe; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; Di Santo, Roberto; Tramontano, Enzo

    2014-10-01

    HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors. PMID:25092689

  4. The lipoxygenase sensor, a new approach in essential fatty acid determination in foods.

    PubMed

    Schoemaker, M; Feldbrügge, R; Gründig, B; Spener, F

    1997-01-01

    Both an enzyme electrode and enzyme column with immobilized lipoxygenase, respectively, were used for the determination of essential fatty acids. The former was applied in a batch system, the latter was part of a fully automated flow injection analysis (FIA)-system. The oxygen consumption due to the lipoxygenase catalysed oxygenation of essential fatty acids was monitored amperometrically. Both systems were compared with regard to linear ranges of the calibration plots, sensitivities, detection limits, apparent Michaelis-Menten constants and lifetimes. The enzyme electrode showed different sensitivities for linoleic and alpha-linolenic acids, the most common essential fatty acids. The reason for this was not a second oxygenation step by lipoxygenase in case of alpha-linolenic acid, but a different dialytic behaviour of the two substrates. Hence, only the FIA-system was used for the determination of these fatty acids in real matrices such as vegetable oils and margarines. In the presence of detergent the triglycerides of the hydrophobic food samples were converted into water soluble glycerol and free fatty acids by a 15 min incubation with a ready to use lipase/esterase-mix, thus avoiding the use of organic solvents for analysis. Results obtained by the enzymatic FIA-system were in excellent agreement with those obtained by standard gas chromatography. PMID:9451797

  5. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed

    Hackney, Kyle J; English, Kirk L

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%-15% of total energy intake (≤1.4 g∙kg-1∙day-1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5-2.0 g∙kg-1∙day-1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  6. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed Central

    Hackney, Kyle J.; English, Kirk L.

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  7. In vitro susceptibility of Brachyspira hyodysenteriae to organic acids and essential oil components

    PubMed Central

    VANDE MAELE, Lien; HEYNDRICKX, Marc; MAES, Dominiek; DE PAUW, Nele; MAHU, Maxime; VERLINDEN, Marc; HAESEBROUCK, Freddy; MARTEL, An; PASMANS, Frank; BOYEN, Filip

    2015-01-01

    The antibacterial potential of organic acids and essential oil components against Brachyspira hyodysenteriae, the causative pathogen of swine dysentery, was evaluated. Minimum inhibitory concentrations (MIC) of 15 compounds were determined at pH 7.2 and pH 6.0, using a broth microdilution assay. In addition, possible synergism was determined. MIC values for the three tested strains were similar. For organic acids, MIC values at pH 6.0 were lower than at pH 7.2. B. hyodysenteriae was most sensitive to cinnamaldehyde and lauric acid, with MIC values <1.5 mM. Most antibacterial effects of binary combinations were additive, however, for thymol and carvacrol, synergism could be observed. In vitro results demonstrate the antibacterial action of certain essential oil components and organic acids against B. hyodysenteriae. PMID:26369432

  8. In vitro susceptibility of Brachyspira hyodysenteriae to organic acids and essential oil components.

    PubMed

    Vande Maele, Lien; Heyndrickx, Marc; Maes, Dominiek; De Pauw, Nele; Mahu, Maxime; Verlinden, Marc; Haesebrouck, Freddy; Martel, An; Pasmans, Frank; Boyen, Filip

    2016-02-01

    The antibacterial potential of organic acids and essential oil components against Brachyspira hyodysenteriae, the causative pathogen of swine dysentery, was evaluated. Minimum inhibitory concentrations (MIC) of 15 compounds were determined at pH 7.2 and pH 6.0, using a broth microdilution assay. In addition, possible synergism was determined. MIC values for the three tested strains were similar. For organic acids, MIC values at pH 6.0 were lower than at pH 7.2. B. hyodysenteriae was most sensitive to cinnamaldehyde and lauric acid, with MIC values <1.5 mM. Most antibacterial effects of binary combinations were additive, however, for thymol and carvacrol, synergism could be observed. In vitro results demonstrate the antibacterial action of certain essential oil components and organic acids against B. hyodysenteriae. PMID:26369432

  9. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  10. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  11. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGESBeta

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  12. The cysteine, total sulfur amino acid, tyrosine, phenylalanine + tyrosine, and non-essential amino acid maintenance requirements of broiler breeders.

    PubMed

    Ekmay, R D; Mei, S J; Sakomura, N K; Coon, C N

    2016-06-01

    Two hundred and fifty Cobb-Vantress broiler breeders were used to determine the maintenance requirement and efficiency of utilization of dietary Cys, Tyr, and non-essential amino acids (AA) in a 21-day experiment. The breeders were fed crystalline amino acid diets containing graded levels of Cys or Tyr representing 0, 10, 20, 30, and 40% of their suggested requirement level with all other amino acids maintained at 40% of their suggested requirement level. To determine the non-essential AA maintenance requirement, graded levels of non-essential AA were provided by glutamic acid to represent 12, 19, 26, 33, and 40% of the ideal level of glutamic acid with all other amino acids maintained at their maintenance requirement level. The total sulfur amino acid (TSAA) and Phe + Tyr requirements were calculated by combining Cys and Tyr results, respectively, with previously determined Met and Phe, respectively. The slope of Cys, Tyr, and non-essential AA accretion regression line indicated that 29% Cys, 24% TSAA, 21% Tyr, 20% Phe + Tyr, and 9% non-essential AA of crystalline amino acids were retained. The Cys requirement for zero protein accretion was calculated to be 30.48 mg/d or 17.006 mg/ kgBW(0.75)/d or 75.426 mg/kgCP/d. The TSAA requirement for zero accretion was calculated to be 132.25 mg/b/d, 71.48 mg/kgBW(0.75)/d, and 307.55 mg/kgCP/d. The Tyr requirement for zero protein accretion was calculated to be 65.907 mg/d or 37.233 mg/ kgBW(0.75)/d or 175.566 mg/kgCP/d. The Phe + Tyr requirement for zero protein accretion was calculated to be 352.18 mg/b/d, 190.37 mg/kgBW(0.75)/d, and 749.33 mg/kgCP/d. The non-essential AA requirement for zero protein accretion was calculated to be 3715.194 mg/d or 2003.155 mg/kgBW(0.75)/d or 9452.954 mg/kgCP/d. PMID:26994191

  13. Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology.

    PubMed

    Canettieri, Eliana Vieira; de Moraes Rocha, George Jackson; de Carvalho, João Andrade; de Almeida e Silva, João Batista

    2007-01-01

    Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars. Hydrolysis can be performed enzymatically and with dilute or concentrate mineral acids. The present study used dilute sulfuric acid as a catalyst for hydrolysis of Eucalyptus grandis residue. The purpose of this paper was to optimize the hydrolysis process in a 1.4 l pilot-scale reactor and investigate the effects of the acid concentration, temperature and residue/acid solution ratio on the hemicellulose removal and consequently on the production of sugars (xylose, glucose and arabinose) as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid). This study was based on a model composition corresponding to a 2(3) orthogonal factorial design and employed the response surface methodology (RSM) to optimize the hydrolysis conditions, aiming to attain maximum xylose extraction from hemicellulose of residue. The considered optimum conditions were: H(2)SO(4) concentration of 0.65%, temperature of 157 degrees C and residue/acid solution ratio of 1/8.6 with a reaction time of 20 min. Under these conditions, 79.6% of the total xylose was removed and the hydrolysate contained 1.65 g/l glucose, 13.65 g/l xylose, 1.55 g/l arabinose, 3.10 g/l acetic acid, 1.23 g/l furfural and 0.20 g/l 5-hydroxymethylfurfural. PMID:16473004

  14. Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding.

    PubMed

    Svensson, B; Clarke, A J; Svendsen, I; Møller, H

    1990-02-22

    Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed. PMID:2108020

  15. Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

    PubMed Central

    Yuan, Shilin; Li, Guiming; Wang, Ying; Gao, Qianqian; Wang, Yizhuo; Cui, Rui

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are

  16. Larvicidal activity of Myrtaceae essential oils and their components against Aedes aegypti, acute toxicity on Daphnia magna, and aqueous residue.

    PubMed

    Park, Hye-Mi; Kim, Junheon; Chang, Kyu-Sik; Kim, Byung-Seok; Yang, Yu-Jung; Kim, Gil-Hah; Shin, Sang-Chul; Park, Il-Kwon

    2011-03-01

    The larvicidal activity of 11 Myrtaceae essential oils and their constituents was evaluated against Aedes aegypti L. Of the 11, Melaleuca linariifolia Sm., Melaleuca dissitiflora F. Muell., Melaleuca quinquenervia (Cav.) S. T. Blake, and Eucalyptus globulus Labill oils at 0.1 mg/ml exhibited > or = 80% larval mortality. At this same concentration, the individual constituents tested, allyl isothiocyanate, alpha-terpinene, p-cymene, (+)-limonene, (-)-limonene, gamma-terpinene, and (E)-nerolidol, resulted in > or = 95% mortality. We also tested the acute toxicity of these four active oils earlier mentioned and their constituents against Daphnia magna Straus. M. linariifolia and allyl isothiocyanate was the most toxic to D. magna. Twodays after treatment, residues of M. dissitiflora, M. linariifolia, M. quinquenervia, and E. globulus oils in water were 55.4, 46.6, 32.4, and 14.8%, respectively. Less than 10% of allyl isothiocyanate, alpha-terpinene, p-cymene, (-)-limonene, (+)-limonene, and gamma-terpinene was detected in the water at 2 d after treatment. Our results indicated that oils and their constituents could easily volatilize in water within a few days after application, thus minimizing their effect on the aqueous ecosystem. Therefore, Myrtaceae essential oils and their constituents could be developed as control agents against mosquito larvae. PMID:21485381

  17. Mycolic Acid Cyclopropanation is Essential for Viability, Drug Resistance, and Cell Wall Integrity of Mycobacterium tuberculosis

    SciTech Connect

    Barkan, Daniel; Liu, Zhen; Sacchettini, James C.; Glickman, Michael S.

    2009-12-01

    Mycobacterium tuberculosis infection remains a major global health problem complicated by escalating rates of antibiotic resistance. Despite the established role of mycolic acid cyclopropane modification in pathogenesis, the feasibility of targeting this enzyme family for antibiotic development is unknown. We show through genetics and chemical biology that mycolic acid methyltransferases are essential for M. tuberculosis viability, cell wall structure, and intrinsic resistance to antibiotics. The tool compound dioctylamine, which we show acts as a substrate mimic, directly inhibits the function of multiple mycolic acid methyltransferases, resulting in loss of cyclopropanation, cell death, loss of acid fastness, and synergistic killing with isoniazid and ciprofloxacin. These results demonstrate that mycolic acid methyltransferases are a promising antibiotic target and that a family of virulence factors can be chemically inhibited with effects not anticipated from studies of each individual enzyme.

  18. Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

    PubMed Central

    Shimamura, A; Nakano, Y J; Mukasa, H; Kuramitsu, H K

    1994-01-01

    The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs. PMID:8050997

  19. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene.

    PubMed

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆⁶desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T₀ and T₁ generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%-0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T₁ generation as well as in immature and mature grains of the T₂ generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%-1.40% (v/v) and 0%-1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  20. Achieving optimal essential fatty acid status in vegetarians: current knowledge and practical implications.

    PubMed

    Davis, Brenda C; Kris-Etherton, Penny M

    2003-09-01

    Although vegetarian diets are generally lower in total fat, saturated fat, and cholesterol than are nonvegetarian diets, they provide comparable levels of essential fatty acids. Vegetarian, especially vegan, diets are relatively low in alpha-linolenic acid (ALA) compared with linoleic acid (LA) and provide little, if any, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Clinical studies suggest that tissue levels of long-chain n-3 fatty acids are depressed in vegetarians, particularly in vegans. n-3 Fatty acids have numerous physiologic benefits, including potent cardioprotective effects. These effects have been demonstrated for ALA as well as EPA and DHA, although the response is generally less for ALA than for EPA and DHA. Conversion of ALA by the body to the more active longer-chain metabolites is inefficient: < 5-10% for EPA and 2-5% for DHA. Thus, total n-3 requirements may be higher for vegetarians than for nonvegetarians, as vegetarians must rely on conversion of ALA to EPA and DHA. Because of the beneficial effects of n-3 fatty acids, it is recommended that vegetarians make dietary changes to optimize n-3 fatty acid status. PMID:12936959

  1. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  2. Identification of important amino acid residues that modulate binding of Escherichia coli GroEL to its various cochaperones.

    PubMed Central

    Klein, G; Georgopoulos, C

    2001-01-01

    Genetic experiments have shown that the GroEL/GroES chaperone machine of Escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including lambda. The virulent bacteriophages T4 and RB49 are independent of the host GroES function, because they encode their own cochaperone proteins, Gp31 and CocO, respectively. E. coli groEL44 mutant bacteria do not form colonies above 42 degrees nor do they propagate bacteriophages lambda, T4, or RB49. We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonies at 43 degrees were due to the presence of an intragenic suppressor mutation. These suppressors define 21 different amino acid substitutions in GroEL, each affecting one of 13 different amino acid residues. All of these amino acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain. All of these intragenic suppressors support bacteriophages lambda, T4, and RB49 growth to various extents in the presence of the groEL44 allele. Since it is known that the GroEL44 mutant protein does not interact effectively with Gp31, the suppressor mutations should enhance cochaperone binding. Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele. PMID:11404317

  3. New charge-bearing amino acid residues that promote β-sheet secondary structure.

    PubMed

    Maynard, Stacy J; Almeida, Aaron M; Yoshimi, Yasuharu; Gellman, Samuel H

    2014-11-26

    Proteinogenic amino acid residues that promote β-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display β-sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also bear an ionizable group in the side chain. PMID:25393077

  4. Uniformly sup 13 C-labeled algal protein used to determine amino acid essentiality in vivo

    SciTech Connect

    Berthold, H.K.; Hachey, D.L.; Reeds, P.J.; Klein, P.D. ); Thomas, O.P. ); Hoeksema, S. )

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with {sup 13}C by growth in an atmosphere of pure {sup 13}CO{sub 2}. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly {sup 13}C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

  5. Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.

    PubMed Central

    Berthold, H K; Hachey, D L; Reeds, P J; Thomas, O P; Hoeksema, S; Klein, P D

    1991-01-01

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo. Images PMID:11607211

  6. Amino Acid Metabolism in Acute Renal Failure: Influence of Intravenous Essential L-Amino Acid Hyperalimentation Therapy

    PubMed Central

    Abel, Ronald M.; Shih, Vivian E.; Abbott, William M.; Beck, Clyde H.; Fischer, Josef E.

    1974-01-01

    A solution of 8 essential I-amino acids and hypertonic dextrose was administered to 5 patients in acute postoperative renal failure in a program of hyperalimentation designed to decrease the patient's catabolic state and to accrue certain metabolic benefits. A sixth patient receiving intravenous glucose alone served as a control. The pretreatment plasma concentrations of amino acids in all 6 patients did not differ significantly from normal; following intravenous essential amino acids at a dose of approximately 12.6 gm/24 hours, no significant elevations out of the normal range of these substances occurred. Since urinary excretion rates did not dramatically increase, urinary loss was excluded as a possible cause for the failure of increase of plasma concentrations. The results suggest that the administration of an intravenous solution of 1-amino acids and hypertonic dextrose is associated with rapid clearance from the blood of these substances and, with a failure of increased urinary excretion, indirect evidence of amino acid utilization for protein synthesis has been obtained. Histidine supplementation in patients with acute renal failure is probably unnecessary based on the lack of significant decreases in histidine concentrations in these patients. PMID:4850497

  7. Effect of co-evolving amino acid residues on topology of phylogenetic trees.

    PubMed

    Sherbakov, D Yu; Triboy, T I

    2007-12-01

    The presence in proteins of amino acid residues that change in concert during evolution is associated with keeping constant the protein spatial structure and functions. As in the case with morphological features, correlated substitutions may become the cause of homoplasies--the independent evolution of identical non-homological adaptations. Our data obtained on model phylogenetic trees and corresponding sets of sequences have shown that the presence of correlated substitutions distorts the results of phylogenetic reconstructions. A method for accounting for co-evolving amino acid residues in phylogenetic analysis is proposed. According to this method, only a single site from the group of correlated amino acid positions should remain, whereas other positions should not be used in further phylogenetic analysis. Simulations performed have shown that replacement on the average of 8% of variable positions in a pair of model sequences by coordinately evolving amino acid residues is able to change the tree topology. The removal of such amino acid residues from sequences before phylogenetic analysis restores the correct topology. PMID:18205620

  8. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  9. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  10. The effects of essential fatty acids preparation in the treatment of intrauterine growth retardation.

    PubMed

    Zhang, L

    1997-10-01

    A treatment of intravenous infusion of glucose, amino acids, and emulsion enriched in essential fatty acids (EFAs), linoleic, and linolenic acids were given to 30 pregnant women with intrauterine growth retardation (IUGR) and 28 non-EFAs treated cases as controls. There was a marked gain in fetal biparietal diameter (BPD) and in the estimated weight of the treated group over the control group. The mean birth weight was significantly different in the two groups. Fetal BPD increased much more in patients treated with EFAs at 28-34 gestational weeks than those at 34 1/7-37 weeks, which indicates that early initiate complement of n-3 and n-6 fatty acids to IUGR mothers may correct pregnancy-induced EFAs deficiency and maternal-fetal malnutrition, which demonstrates a fetal catch-up of growth in the brain and the whole body. PMID:9394162

  11. Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

    PubMed

    Xiong, Z; Farilla, L; Guo, J; McLachlan, S; Rapoport, B

    2001-03-01

    Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741. PMID:11327613

  12. Mechanisms of lipid malabsorption in Cystic Fibrosis: the impact of essential fatty acids deficiency

    PubMed Central

    Peretti, N; Marcil, V; Drouin, E; Levy, E

    2005-01-01

    Transport mechanisms, whereby alimentary lipids are digested and packaged into small emulsion particles that enter intestinal cells to be translocated to the plasma in the form of chylomicrons, are impaired in cystic fibrosis. The purpose of this paper is to focus on defects that are related to intraluminal and intracellular events in this life-limiting genetic disorder. Specific evidence is presented to highlight the relationship between fat malabsorption and essential fatty acid deficiency commonly found in patients with cystic fibrosis that are often related to the genotype. Given the interdependency of pulmonary disease, pancreatic insufficiency and nutritional status, greater attention should be paid to the optimal correction of fat malabsorption and essential fatty acid deficiency in order to improve the quality of life and extend the life span of patients with cystic fibrosis. PMID:15869703

  13. Search for conserved amino acid residues of the [Formula: see text]-crystallin proteins of vertebrates.

    PubMed

    Shiliaev, Nikita G; Selivanova, Olga M; Galzitskaya, Oxana V

    2016-04-01

    [Formula: see text]-crystallin is the major eye lens protein and a member of the small heat-shock protein (sHsp) family. [Formula: see text]-crystallins have been shown to support lens clarity by preventing the aggregation of lens proteins. We performed the bioinformatics analysis of [Formula: see text]-crystallin sequences from vertebrates to find conserved amino acid residues as the three-dimensional (3D) structure of [Formula: see text]-crystallin is not identified yet. We are the first who demonstrated that the N-terminal region is conservative along with the central domain for vertebrate organisms. We have found that there is correlation between the conserved and structured regions. Moreover, amyloidogenic regions also correspond to the structured regions. We analyzed the amino acid composition of [Formula: see text]-crystallin A and B chains. Analyzing the occurrence of each individual amino acid residue, we have found that such amino acid residues as leucine, serine, lysine, proline, phenylalanine, histidine, isoleucine, glutamic acid, and valine change their content simultaneously in A and B chains in different classes of vertebrates. Aromatic amino acids occur more often in [Formula: see text]-crystallins from vertebrates than on the average in proteins among 17 animal proteomes. We obtained that the identity between A and B chains in the mammalian group is 0.35, which is lower than the published 0.60. PMID:26972563

  14. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase.

    PubMed

    Matsuzawa, Tomohiko; Saito, Yuji; Yaoi, Katsuro

    2014-05-01

    Unlike endo-dissociative-xyloglucanases, Paenibacillus XEG74 is an endo-processive xyloglucanase that contains four unique tryptophan residues in the negative subsites (W61 and W64) and the positive subsites (W318 and W319), as indicated by three-dimensional homology modelling. Selective replacement of the positive subsite residues with alanine mutations reduced the degree of processive activity and resulted in the more endo-dissociative-activity. The results showed that W318 and W319, which are found in the positive subsites, are essential for processive degradation and are responsible for maintaining binding interactions with xyloglucan polysaccharide through a stacking effect. PMID:24657616

  15. Protein reactivity with singlet oxygen: Influence of the solvent exposure of the reactive amino acid residues.

    PubMed

    Sjöberg, Béatrice; Foley, Sarah; Staicu, Angela; Pascu, Alexandru; Pascu, Mihail; Enescu, Mironel

    2016-06-01

    The singlet oxygen quenching rate constants were measured for three model proteins, bovine serum albumin, β-lactoglobulin and lysozyme. The results were analyzed by comparing them with the corresponding singlet oxygen quenching rate constants for a series of tripeptides with the basic formula GlyAAGly where the central amino acid (AA) was the oxidizable amino acid, tryptophan, tyrosine, methionine and histidine. It was found that the reaction rate constant in proteins can be satisfactorily modelled by the sum of the individual contributions of the oxidizable AA residues corrected for the solvent accessible surface area (SASA) effects. The best results were obtained when the SASA of the AA residues were determined by averaging over molecular dynamics simulated trajectories of the proteins. The limits of this geometrical correction of the AA residue reactivity are also discussed. PMID:27045278

  16. Isoelectric Point, Electric Charge, and Nomenclature of the Acid-Base Residues of Proteins

    ERIC Educational Resources Information Center

    Maldonado, Andres A.; Ribeiro, Joao M.; Sillero, Antonio

    2010-01-01

    The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. (Contains 8 figures and 4 tables.)

  17. Identification of a key residue in Kv7.1 potassium channel essential for sensing external potassium ions

    PubMed Central

    Wang, Wenying; Flores, Maria Cristina Perez; Sihn, Choong-Ryoul; Kim, Hyo Jeong; Zhang, Yinuo; Doyle, Karen J.; Chiamvimonvat, Nipavan

    2015-01-01

    Kv7.1 voltage-gated K+ (Kv) channels are present in the apical membranes of marginal cells of the stria vascularis of the inner ear, where they mediate K+ efflux into the scala media (cochlear duct) of the cochlea. As such, they are exposed to the K+-rich (∼150 mM of external K+ (K+e)) environment of the endolymph. Previous studies have shown that Kv7.1 currents are substantially suppressed by high K+e (independent of the effects of altering the electrochemical gradient). However, the molecular basis for this inhibition, which is believed to involve stabilization of an inactivated state, remains unclear. Using sequence alignment of S5-pore linkers of several Kv channels, we identified a key residue, E290, found in only a few Kv channels including Kv7.1. We used substituted cysteine accessibility methods and patch-clamp analysis to provide evidence that the ability of Kv7.1 to sense K+e depends on E290, and that the charge at this position is essential for Kv7.1’s K+e sensitivity. We propose that Kv7.1 may use this feedback mechanism to maintain the magnitude of the endocochlear potential, which boosts the driving force to generate the receptor potential of hair cells. The implications of our findings transcend the auditory system; mutations at this position also result in long QT syndrome in the heart. PMID:25712016

  18. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

    PubMed Central

    Sawada, Kazutaka

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation. PMID:26839744

  19. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation.

    PubMed

    Sawada, Kazutaka; Kitagaki, Hiroshi

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation. PMID:26839744

  20. Combined antimicrobial effect of oregano essential oil and caprylic acid in minced beef.

    PubMed

    Hulankova, Radka; Borilova, Gabriela; Steinhauserova, Iva

    2013-10-01

    Oregano essential oil (OEO) and caprylic acid (CA) are highly aromatic natural antimicrobials with limited individual application in food. We proved their combined additive effect when used in meat. Application of 0.5% CA and 0.2% OEO (v/w) with 0.1% of citric acid in vacuum packed minced beef inoculated with Listeria monocytogenes at a concentration of 5 log cells/g reduced counts of lactic acid bacteria by 1.5 log CFU/g and counts of psychrotrophic bacteria and L. monocytogenes by more than 2.5 log CFU/g at the end of storage at 3°C for 10 days. In sensory evaluation the samples with OEO showed during the whole experiment statistically better scores than control, whereas the samples treated with CA showed worse colour attributes. PMID:23743028

  1. Dietary Deficiency of Essential Amino Acids Rapidly Induces Cessation of the Rat Estrous Cycle

    PubMed Central

    Bannai, Makoto; Ichimaru, Toru; Nakano, Sayako; Murata, Takuya; Higuchi, Takashi; Takahashi, Michio

    2011-01-01

    Reproductive functions are regulated by the sophisticated coordination between the neuronal and endocrine systems and are sustained by a proper nutritional environment. Female reproductive function is vulnerable to effects from dietary restrictions, suggesting a transient adaptation that prioritizes individual survival over reproduction until a possible future opportunity for satiation. This adaptation could also partially explain the existence of amenorrhea in women with anorexia nervosa. Because amino acid nutritional conditions other than caloric restriction uniquely alters amino acid metabolism and affect the hormonal levels of organisms, we hypothesized that the supply of essential amino acids in the diet plays a pivotal role in the maintenance of the female reproductive system. To test this hypothesis, we examined ovulatory cyclicity in female rats under diets that were deficient in threonine, lysine, tryptophan, methionine or valine. Ovulatory cyclicity was monitored by daily cytological evaluations of vaginal smears. After continuous feeding of the deficient diet, a persistent diestrus or anovulatory state was induced most quickly by the valine-deficient diet and most slowly by the lysine-deficient diet. A decline in the systemic insulin-like growth factor 1 level was associated with a dietary amino acid deficiency. Furthermore, a paired group of rats that were fed an isocaloric diet with balanced amino acids maintained normal estrous cyclicity. These disturbances of the estrous cycle by amino acid deficiency were quickly reversed by the consumption of a normal diet. The continuous anovulatory state in this study is not attributable to a decrease in caloric intake but to an imbalance in the dietary amino acid composition. With a shortage of well-balanced amino acid sources, reproduction becomes risky for both the mother and the fetus. It could be viewed as an adaptation to the diet, diverting resources away from reproduction and reallocating them to

  2. Characteristics of lactic acid bacteria isolates and their effect on silage fermentation of fruit residues.

    PubMed

    Yang, Jinsong; Tan, Haisheng; Cai, Yimin

    2016-07-01

    The natural lactic acid bacteria (LAB) population, chemical composition, and silage fermentation of fruit residues were studied. Eighty-two strains of LAB were isolated from fruit residues such as banana leaf and stem, pineapple peel, and papaya peel. All strains were gram-positive and catalase-negative bacteria, and they were divided into 7 groups (A-G) according to morphological and biochemical characters. Strains in groups A to F were rods, and group G was cocci. Group F produced gas from glucose; other groups did not. Groups A to C and F formed dl-lactic acid, whereas groups D, E, and G formed l-lactic acid. Based on the 16S rRNA gene sequence and DNA-DNA hybridization analysis, groups A to G strains were identified as Lactobacillus plantarum (54.9% of the total isolates), Lactobacillus paraplantarum (3.6%), Lactobacillus nagelii (8.5%), Lactobacillus perolens (4.9%), Lactobacillus casei (11.0%), Lactobacillus fermentum (9.8%), and Enterococcus gallinarum (7.3%), respectively. Lactobacillus plantarum and Lactobacillus casei are the most frequently isolated from fruit residues as a dominant species, and they could grow at a lower pH conditions and produce more lactic acid than other isolates. Pineapple and papaya peels contained higher crude protein (11.5-13.8%) and water-soluble carbohydrate (16.8-22.4%), but lower acid detergent fiber contents (21.2 to 26.4%) than banana stems and leaves (8.2% crude protein, 42.8% acid detergent fiber, and 5.1% water-soluble carbohydrate). Compared with banana stem and leaf silages, the pineapple and papaya peel silages were well preserved with a lower pH and higher lactate content. The study suggests that the fruit residues contain excellent LAB species and abundant feed nutrients, and that they can be preserved as silage to be potential food resources for livestock. PMID:27108171

  3. Anthelmintic efficacy of cinnamaldehyde and cinnamic acid from cortex cinnamon essential oil against Dactylogyrus intermedius.

    PubMed

    Ling, Fei; Jiang, Chao; Liu, Guanglu; Li, Mingshuang; Wang, Gaoxue

    2015-12-01

    Utilization of chemical pesticide to control monogenean diseases is often restricted in many countries due to the development of pesticide resistance and concerns of chemical residues and environmental contamination. Thus, the use of antiparasitic agents from plants has been explored as a possible way for controlling monogenean infections. Extracts from Cinnamomum cassia were investigated under in vivo conditions against Dactylogyrus intermedius in goldfish. The two bioactive compounds, cinnamaldehyde and cinnamic acid, were identified using nuclear magnetic resonance and electrospray ionization mass spectrometry. The 48 h median effective concentrations (EC(50)) for these compounds against D. intermedius were 0·57 and 6·32 mg L(-1), respectively. The LD(50) of cinnamaldehyde and cinnamic acid were 13·34 and 59·66 mg L(-1) to goldfish in 48 h acute toxicity tests, respectively. These data confirm that cinnamaldehyde is effective against D. intermedius, and the cinnamaldehyde exhibits potential for the development of a candidate antiparasitic agent. PMID:26442478

  4. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  5. Intramolecular cyclization of aspartic acid residues assisted by three water molecules: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Takahashi, Ohgi; Kirikoshi, Ryota

    2014-01-01

    Aspartic acid (Asp) residues in peptides and proteins (l-Asp) are known to undergo spontaneous nonenzymatic reactions to form l-β-Asp, d-Asp, and d-β-Asp residues. The formation of these abnormal Asp residues in proteins may affect their three-dimensional structures and hence their properties and functions. Indeed, the reactions have been thought to contribute to aging and pathologies. Most of the above reactions of the l-Asp residues proceed via a cyclic succinimide intermediate. In this paper, a novel three-water-assisted mechanism is proposed for cyclization of an Asp residue (forming a gem-diol precursor of the succinimide) by the B3LYP/6-31 + G(d,p) density functional theory calculations carried out for an Asp-containing model compound (Ace-Asp-Nme, where Ace = acetyl and Nme = NHCH3). The three water molecules act as catalysts by mediating ‘long-range’ proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form (iminolization). Then, reorientation of a water molecule and a conformational change occur successively, followed by the nucleophilic attack of the iminol nitrogen on the carboxyl carbon of the Asp side chain to form the gem-diol species. A satisfactory agreement was obtained between the calculated and experimental energetics.

  6. A novel sono-assisted acid pretreatment of chili post harvest residue for bioethanol production.

    PubMed

    Sindhu, Raveendran; Binod, Parameswaran; Pandey, Ashok

    2016-08-01

    The objective of the present study was to develop a sono-assisted acid pretreatment strategy for the effective removal of lignin and hemicelluloses and to improve the sugar yield from chili post harvest residue. Operational parameters that affect the pretreatment efficiency were studied and optimized. Inhibitor analysis of the hydrolyzate revealed that major fermentation inhibitors like furfural, hydroxymethyl furfural and organic acids like citric acid, succinic acid and propionic acid were absent. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method yielded 0.465g/g of reducing sugars on enzymatic hydrolysis. Fermentation of the non-detoxified hydrolysate yielded 2.14% of bioethanol with a fermentation efficiency of 71.03%. PMID:26949055

  7. Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry*

    PubMed Central

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E.

    2011-01-01

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  8. Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 contain reduced cysteine residues essential for virus entry.

    PubMed

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E

    2011-09-16

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  9. Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

    PubMed Central

    Boudko, Dmitri Y.

    2012-01-01

    Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B0 transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B0-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes

  10. THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.

    PubMed

    Cox, Jonathan A G; Abrahams, Katherine A; Alemparte, Carlos; Ghidelli-Disse, Sonja; Rullas, Joaquín; Angulo-Barturen, Iñigo; Singh, Albel; Gurcha, Sudagar S; Nataraj, Vijayashankar; Bethell, Stephen; Remuiñán, Modesto J; Encinas, Lourdes; Jervis, Peter J; Cammack, Nicholas C; Bhatt, Apoorva; Kruse, Ulrich; Bantscheff, Marcus; Fütterer, Klaus; Barros, David; Ballell, Lluis; Drewes, Gerard; Besra, Gurdyal S

    2016-01-01

    Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors. PMID:27571973

  11. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria

    PubMed Central

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-01-01

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme. PMID:26628098

  12. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria.

    PubMed

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-01-01

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme. PMID:26628098

  13. Chlorogenic acid content, essential oil compositions, and in vitro antioxidant activities of Chromolaena odorata leaves.

    PubMed

    Pitakpawasutthi, Yamon; Thitikornpong, Worathat; Palanuvej, Chanida; Ruangrungsi, Nijsiri

    2016-01-01

    Chromolaena odorata (L.) R. M. King and H. Rob. is a Thai medicinal plant used for the treatment of wounds, rashes, diabetes, and insect repellent. The leaves of C. odorata were collected from 10 different sources throughout Thailand. The chemical constituents of essential oils were hydro-distilled from the leaves and were analyzed by gas chromatography-mass spectrometry. Chlorogenic acid contents were determined by thin-layer chromatography (TLC) - densitometry with winCATS software and TLC image analysis with ImageJ software. The TLC plate was developed in the mobile phase that consisted of ethyl acetate:water:formic acid (17:3:2). Antioxidant activities were examined by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging and β-carotene bleaching assays. C. odorata essential oil has shown the major components of pregeijerene, dauca-5, 8-diene, (E)-caryophyllene, β-pinene, and α-pinene. The chlorogenic acid content of C. odorata leaves was determined by TLC-densitometry and TLC image analysis. Results have shown that TLC-densitometry and TLC image analysis method were not statistically significantly different. DPPH radical scavenging and β-carotene bleaching assays of ethanolic extract of C. odorata leaves showed its antioxidant potential. PMID:27144150

  14. Chlorogenic acid content, essential oil compositions, and in vitro antioxidant activities of Chromolaena odorata leaves

    PubMed Central

    Pitakpawasutthi, Yamon; Thitikornpong, Worathat; Palanuvej, Chanida; Ruangrungsi, Nijsiri

    2016-01-01

    Chromolaena odorata (L.) R. M. King and H. Rob. is a Thai medicinal plant used for the treatment of wounds, rashes, diabetes, and insect repellent. The leaves of C. odorata were collected from 10 different sources throughout Thailand. The chemical constituents of essential oils were hydro-distilled from the leaves and were analyzed by gas chromatography-mass spectrometry. Chlorogenic acid contents were determined by thin-layer chromatography (TLC) - densitometry with winCATS software and TLC image analysis with ImageJ software. The TLC plate was developed in the mobile phase that consisted of ethyl acetate:water:formic acid (17:3:2). Antioxidant activities were examined by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging and β-carotene bleaching assays. C. odorata essential oil has shown the major components of pregeijerene, dauca-5, 8-diene, (E)-caryophyllene, β-pinene, and α-pinene. The chlorogenic acid content of C. odorata leaves was determined by TLC-densitometry and TLC image analysis. Results have shown that TLC-densitometry and TLC image analysis method were not statistically significantly different. DPPH radical scavenging and β-carotene bleaching assays of ethanolic extract of C. odorata leaves showed its antioxidant potential. PMID:27144150

  15. The Role of Essential Fatty Acids in Anorexia Nervosa and Obesity.

    PubMed

    Yehuda, Shlomo; Rabinovitz, Sharon

    2016-09-01

    The two basic questions in food intake study are what we eat, and how much do we eat. Most research is directed toward the control of how much is eaten. This is likely the result of the increased number of individuals with eating disorders in the Western world. Feeding behavior is highly complex, and is controlled by many psychological, physiological, biochemical, and immunological factors. The aim of this review is to clarify the involvement of fatty acids in eating disorders such as anorexia and binge eating disorder. The review will describe the modified fatty acid profile observed in individuals with anorexia or binge eating disorder, and discuss on what factors fatty acids can exert beneficial effects. In addition, the differences and similarities between anorexia and binge eating disorder will be discussed. We suggest that beneficial effects of essential fatty acids on both anorexia and binge eating disorder can be explained by the stabilizing effect of those fatty acids on the neuronal membrane fluidity index. PMID:26068122

  16. Dietary essential fatty acids and gender-specific behavioral responses in cranially irradiated rats

    PubMed Central

    Elkin, T David; Wollan, Michael O; Anderson, Stacy L; Gaston, Robert; Meyer, William; Fuemmeler, Bernard F; Holloway, Frank A; Martin, Rex E

    2006-01-01

    Specific memory deficits, reduced intellectual processing speed, and a variety of social and behavioral problems have been implicated as long-term effects of cranial radiation therapy (CRT). These deficits are thought to be related to changes in brain cytology and structure associated with microvascular aberrations. N-3 fatty acids may serve as protectants in pediatric patients who receive CRT for brain tumors. Timed-pregnant rat dams were fed one of four diets that were identical in all respects, except for their essential fatty acid content. The dams were placed on these diets at the beginning of the third trimester of gestation and their pups remained on them throughout the study. The rats’ behavioral response as judged by acoustic startle response (ASR) and neurocognitive response (performance in a radial maze, RM) were evaluated in relation to diet, gender, and CRT. The following hypotheses were tested: (1) female rats will show greater CRT-induced neurocognitive and behavioral deficits; (2) dietary n-3 fatty acids will diminish CRT-induced neurocognitive and behavioral deficits; (3) gender-specific differences would be dampened by n-3 fatty acids in the diet. All three hypotheses were partially supported. These findings are discussed in light of the potential neuroprotective effects of n-3 fatty acids. PMID:19412484

  17. Nutrition in brain development and aging: role of essential fatty acids.

    PubMed

    Uauy, Ricardo; Dangour, Alan D

    2006-05-01

    The essential fatty acids (EFAs), particularly the n-3 long-chain polyunsaturated fatty acids (LCPs), are important for brain development during both the fetal and postnatal period. They are also increasingly seen to be of value in limiting the cognitive decline during aging. EFA deficiency was first shown over 75 years ago, but the more subtle effects of the n-3 fatty acids in terms of skin changes, a poor response to linoleic acid supplementation, abnormal visual function, and peripheral neuropathy were only discovered later. Both n-3 and n-6 LCPs play important roles in neuronal growth, development of synaptic processing of neural cell interaction, and expression of genes regulating cell differentiation and growth. The fetus and placenta are dependent on maternal EFA supply for their growth and development, with docosahexaenomic acid (DHA)-supplemented infants showing significantly greater mental and psychomotor development scores (breast-fed children do even better). Dietary DHA is needed for the optimum functional maturation of the retina and visual cortex, with visual acuity and mental development seemingly improved by extra DHA. Aging is also associated with decreased brain levels of DHA: fish consumption is associated with decreased risk of dementia and Alzheimer's disease, and the reported daily use of fish-oil supplements has been linked to improved cognitive function scores, but confirmation of these effects is needed. PMID:16770950

  18. The cyst wall of Colpoda steinii. A substance rich in glutamic acid residues

    PubMed Central

    Tibbs, J.

    1966-01-01

    1. The cyst wall of Colpoda steinii has been isolated and its chemical nature examined. It had a nitrogen content 13·9±0·2% (s.d.) and an ash 8·6±1·6% (s.d.). After lipid and hot-acid extraction there was a variable residual phosphorus of 0·19–0·64%. The protein nature, indicated by infrared and ultraviolet absorption, was confirmed when 100μg. of hydrolysed wall gave a ninhydrin colour equivalent to that given by 0·88–1·01μmoles of glycine. Hexosamine, hexose, pentose, lipid and dipicolinic acid were absent. 2. Paper chromatography of hydrolysates, besides showing the presence of the usual protein amino acids and three unidentified ninhydrin-reacting spots, indicated the presence of large amounts of glutamic acid. Estimated by chromatography, the amount present was 52·9±0·6 (s.d.) g./100g. of ash-free wall; manometric estimation of l-glutamic acid with l-glutamate 1-carboxy-lyase gave 46·5±0·9 (s.d.) g./100g. 3. Free carboxyl groups were estimated by titration as 0·159±0·011 (s.d.) mole/100g. and those present as amide as 0·154±0·004 (s.d.) mole/100g., and the total was compared with the dicarboxylic acid content 0·360±0·010 (s.d.) mole/100g. 4. After treatment with 98% formic acid 25–30% of the wall material could be extracted by 0·05m-sodium carbonate solution (extract 1); after treatment of the residue with performic acid a further 62–63% based on the original weight could be extracted by 0·05m-sodium carbonate (extract 2). 5. The average values found for the glutamic acid contents were 21·7g./100g. for extract 1 and 58·0g./100g. for extract 2. The cysteic acid content of whole oxidized wall was about 5·8g./100g. and of extract 2 also about 5·8g./100g. The glutamic acid and cysteic acid contents of the final residue were also investigated. 6. The significance of these extraction experiments in relation to the wall structure is discussed. ImagesPlate 1. PMID:4957913

  19. Essentials in the diagnosis of acid-base disorders and their high altitude application.

    PubMed

    Paulev, P E; Zubieta-Calleja, G R

    2005-09-01

    This report describes the historical development in the clinical application of chemical variables for the interpretation of acid-base disturbances. The pH concept was already introduced in 1909. Following World War II, disagreements concerning the definition of acids and bases occurred, and since then two strategies have been competing. Danish scientists in 1923 defined an acid as a substance able to give off a proton at a given pH, and a base as a substance that could bind a proton, whereas the North American Singer-Hasting school in 1948 defined acids as strong non-buffer anions and bases as non-buffer cations. As a consequence of this last definition, electrolyte disturbances were mixed up with real acid-base disorders and the variable, strong ion difference (SID), was introduced as a measure of non-respiratory acid-base disturbances. However, the SID concept is only an empirical approximation. In contrast, the Astrup/Siggaard-Andersen school of scientists, using computer strategies and the Acid-base Chart, has made diagnosis of acid-base disorders possible at a glance on the Chart, when the data are considered in context with the clinical development. Siggaard-Andersen introduced Base Excess (BE) or Standard Base Excess (SBE) in the extracellular fluid volume (ECF), extended to include the red cell volume (eECF), as a measure of metabolic acid-base disturbances and recently replaced it by the term Concentration of Titratable Hydrogen Ion (ctH). These two concepts (SBE and ctH) represent the same concentration difference, but with opposite signs. Three charts modified from the Siggaard-Andersen Acid-Base Chart are presented for use at low, medium and high altitudes of 2500 m, 3500 m, and 4000 m, respectively. In this context, the authors suggest the use of Titratable Hydrogen Ion concentration Difference (THID) in the extended extracellular fluid volume, finding it efficient and better than any other determination of the metabolic component in acid

  20. Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

    PubMed Central

    Gajardo, R; Vende, P; Poncet, D; Cohen, J

    1997-01-01

    Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7. PMID:9032355

  1. Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

    PubMed Central

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

    2015-01-01

    Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism. PMID:25588215

  2. Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation

    PubMed Central

    Michel, Detlef; Kramer, Silke; Höhn, Simone; Schaarschmidt, Peter; Wunderlich, Kirsten; Mertens, Thomas

    1999-01-01

    Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation. PMID:10482650

  3. Antimicrobial Efficacy of an Array of Essential Oils Against Lactic Acid Bacteria.

    PubMed

    Dunn, Laurel L; Davidson, P Michael; Critzer, Faith J

    2016-02-01

    The essential oils of clove bud, cinnamon bark and thyme, and their individual compounds including allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde, cinnamic acid, eugenol, and thymol were initially assessed for antimicrobial activity against 9 lactic acid bacteria (LAB) species. Carvacrol and thymol were the most inhibitory with MICs of 0.1% (v/v and w/v, respectively). Cinnamaldehyde, cinnamon bark oil, clove bud oil, eugenol, and thyme oil were moderately inhibitive (MICs = 0.2% v/v), while cinnamic acid required a concentration of 0.5% (w/v). AIT was not effective with MICs in excess of concentrations tested (0.75% v/v). The bactericidal capability of the oil components carvacrol, cinnamaldehyde, eugenol, and thymol were further examined against Pediococcus acidilactici, Lactobacillus buchneri, and Leuconostoc citrovorum. Thymol at 0.1% (w/v) was bactericidal against L. citrovorum (>4-log reduction), but resulted in a 2-log CFU/mL reduction against L. buchneri and P. acidilactici. Cinnamaldehyde at 0.2% to 0.25% (v/v) was effective against L. citrovorum, L. buchneri, and P. acidilactici, resulting in a >2-log reduction. All 3 organisms were susceptible to 0.2% carvacrol with >3-log reduction observed after exposure for 6 h. Eugenol was the least effective. Concentrations of 0.2% and 0.25% (v/v) were needed to achieve an initial reduction in population, >3-log CFU/mL after 6 h exposure. However, at 0.2%, P. acidilactici and L. buchneri recovered to initial populations in 48 to 72 h. Results indicate essential oils have the capacity to inactivate LAB that are commonly associated with spoilage of shelf stable low-acid foods. PMID:26749216

  4. Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues

    SciTech Connect

    Pande, C.S.; Pelzig, M.; Glass, J.D.

    1980-02-01

    Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonsulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents.The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.

  5. The periplasmic alpha-carbonic anhydrase activity of Helicobacter pylori is essential for acid acclimation.

    PubMed

    Marcus, Elizabeth A; Moshfegh, Amiel P; Sachs, George; Scott, David R

    2005-01-01

    The role of the periplasmic alpha-carbonic anhydrase (alpha-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since alpha-CA catalyzes the conversion of CO2 to HCO3-, the role of CO2 in periplasmic buffering was studied using an alpha-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that alpha-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the alpha-CA knockout. In the mutant or in the presence of acetazolamide, there was an approximately 3 log10 decrease in acid survival. In acid, absence of alpha-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the alpha-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of alpha-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3- by the periplasmic alpha-CA. PMID:15629943

  6. Stable isotope studies reveal pathways for the incorporation of non-essential amino acids in Acyrthosiphon pisum (pea aphids).

    PubMed

    Haribal, Meena; Jander, Georg

    2015-12-01

    Plant roots incorporate inorganic nitrogen into the amino acids glutamine, glutamic acid, asparagine and aspartic acid, which together serve as the primary metabolites of nitrogen transport to other tissues. Given the preponderance of these four amino acids, phloem sap is a nutritionally unbalanced diet for phloem-feeding insects. Therefore, aphids and other phloem feeders typically rely on microbial symbionts for the synthesis of essential amino acids. To investigate the metabolism of the four main transport amino acids by the pea aphid (Acyrthosiphon pisum), and its Buchnera aphidicola endosymbionts, aphids were fed defined diets with stable isotope-labeled glutamine, glutamic acid, asparagine or aspartic acid (U-(13)C, U-(15)N; U-(15)N; α-(15)N; or γ-(15)N). The metabolic fate of the dietary (15)N and (13)C was traced using gas chromatography-mass spectrometry (GC-MS). Nitrogen was the major contributor to the observed amino acid isotopomers with one additional unit mass (M+1). However, there was differential incorporation, with the amine nitrogen of asparagine being incorporated into other amino acids more efficiently than the amide nitrogen. Higher isotopomers (M+2, M+3 and M+4) indicated the incorporation of varying numbers of (13)C atoms into essential amino acids. GC-MS assays also showed that, even with an excess of dietary labeled glutamine, glutamic acid, asparagine or aspartic acid, the overall content of these amino acids in aphid bodies was mostly the product of catabolism of dietary amino acids and subsequent re-synthesis within the aphids. Thus, these predominant dietary amino acids are not passed directly to Buchnera endosymbionts for synthesis of essential amino acids, but are rather are produced de novo, most likely by endogenous aphid enzymes. PMID:26632455

  7. Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.

    PubMed

    Herr, F M; Matarese, V; Bernlohr, D A; Storch, J

    1995-09-19

    The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7547918

  8. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

  9. Biochemical evaluation of borage (Borago officinalis) rosette leaves through their essential oil and fatty acid composition.

    PubMed

    Mhamdi, Baya; Aidi Wannes, Wissem; Marzouk, Brahim

    2007-06-01

    Borago officinalis rosette leaves were sampled in the region of Amdoun (Tunisia) during different stages of their development. Essential oil contents varied from 0.01% to 0.13% respectively in young and adult leaves. Twenty three volatile compounds were identified. Hydrocarbons, mainly represented by nonadecane (29.8%), tetracosane (11.3%) and heptacosane (4.7%), constituted the major class in the young leaves (45.8%), followed by aldehydes (22.4%). The percentages of these two classes decreased to reach respectively 15% and 8.1% in adult leaves in favour of alcohols (57.9%) where cis-3-hexenol (29.6%) and hexanol (14.5%) were the main compounds. Total fatty acids amounts increased from 5.03 mg/g DW in young leaves to 32.23 mg/g DW in adult ones. The predominant fatty acids were alpha-linolenic (C18:3 n-3), stearidonic (C18:4 n-3), gamma-linolenic (C18:3 n-6), palmitic (C16: 0) and linoleic (C18:2 n-6) acids. PMID:17722661

  10. Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore midgut

    PubMed Central

    Chen, Hui; Wilkerson, Curtis G.; Kuchar, Jason A.; Phinney, Brett S.; Howe, Gregg A.

    2005-01-01

    The plant hormone jasmonic acid (JA) activates host defense responses against a broad spectrum of herbivores. Although it is well established that JA controls the expression of a large set of target genes in response to tissue damage, very few gene products have been shown to play a direct role in reducing herbivore performance. To test the hypothesis that JA-inducible proteins (JIPs) thwart attack by disrupting digestive processes in the insect gut, we used a MS-based approach to identify host proteins that accumulate in the midgut of Manduca sexta larvae reared on tomato (Solanum lycopersicum) plants. We show that two JIPs, arginase and threonine deaminase (TD), act in the M. sexta midgut to catabolize the essential amino acids Arg and Thr, respectively. Transgenic plants that overexpress arginase were more resistant to M. sexta larvae, and this effect was correlated with reduced levels of midgut Arg. We present evidence indicating that the ability of TD to degrade Thr in the midgut is enhanced by herbivore-induced proteolytic removal of the enzyme's C-terminal regulatory domain, which confers negative feedback regulation by isoleucine in planta. Our results demonstrate that the JA signaling pathway strongly influences the midgut protein content of phytophagous insects and support the hypothesis that catabolism of amino acids in the insect digestive tract by host enzymes plays a role in plant protection against herbivores. PMID:16357201

  11. Essential role of docosahexaenoic acid towards development of a smarter brain.

    PubMed

    Gharami, Kusumika; Das, Moitreyi; Das, Sumantra

    2015-10-01

    Evolution of the high order brain function in humans can be attributed to intake of poly unsaturated fatty acids (PUFAs) of which the ω-3 fatty acid, docosahexaenoic acid (DHA) has special significance. DHA is abundantly present in the human brain and is an essential requirement in every step of brain development like neural cell proliferation, migration, differentiation, synaptogenesis etc. The multiple double bonds and unique structure allow DHA to impart special membrane characteristics for effective cell signaling. Evidences indicate that DHA accumulate in areas of the brain associated with learning and memory. Many development disorders like dyslexia, autism spectrum disorder, attention deficit hyperactivity disorder, schizophrenia etc. are causally related to decreased level of DHA. The review discusses the various reports of DHA in these areas for a better understanding of the role of DHA in overall brain development. Studies involving laboratory animals and clinical findings in cases as well as during trials have been taken into consideration. Additionally the currently available dietary source of DHA for supplementation as nutraceutics with general caution for overuse has been examined. PMID:26321578

  12. Unaffected Arm Muscle Hypercatabolism in Dysphagic Subacute Stroke Patients: The Effects of Essential Amino Acid Supplementation

    PubMed Central

    Aquilani, Roberto; Boselli, Mirella; D'Antona, Giuseppe; Baiardi, Paola; Boschi, Federica; Viglio, Simona; Iadarola, Paolo; Pasini, Evasio; Barbieri, Annalisa; Dossena, Maurizia; Bongiorno, Andria Innocenza; Verri, Manuela

    2014-01-01

    Alterations in muscle protein turnover of the unaffected side of stroke patients could contribute to physical disability. We investigated whether hypercatabolic activity occurred in unaffected arm muscle and whether supplemented essential amino acids (EAAs) could limit muscle hypercatabolism (MH). Thirty-eight dysphagic subacute stroke subjects (<3 months after acute event) (29 males + 9 females; 69.7 ± 11.4 yrs) were enrolled and randomized to receive 8 g/day EAAs (n = 19; EAA group) or isocaloric placebo (maltodextrin; n = 19, Plac group). Before randomization, all patients had their arterial (A) and venous (V) amino acids measured and muscle (A − V) differences calculated in the unaffected arm. Eight matched and healthy subjects served as controls. When compared to healthy controls, the entire stroke population showed significant muscle release (= negative value A − V) of the amino acid phenylalanine (phenyl-) indicating a prevalence of MH. Moreover, randomized EAA and Plac groups had similar rates of MH. After 38 days from the start of the protocol, the EAA group but not the Plac group had MH converted to balanced protein turnover or anabolic activity. We concluded that muscle protein metabolism of the unaffected arm of dysphagic subacute stroke individuals could be characterized by MH which can be corrected by supplemented EAAs. PMID:25431770

  13. Inhibition of nocturnal acidity is important but not essential for duodenal ulcer healing.

    PubMed Central

    Bianchi Porro, G; Parente, F; Sangaletti, O

    1990-01-01

    We have determined the relative importance of day and night time gastric acid inhibition for duodenal ulcer healing by comparing the anti-ulcer efficacy of a single morning with that of a single bedtime dose of ranitidine. One hundred and thirty patients with active duodenal ulcer were randomly assigned to a double-blind therapy with ranitidine 300 mg at 8 am or the same dose at 10 pm for up to eight weeks. The antisecretory effects of these regimens were also assessed by 24 h intragastric pH monitoring in 18 of these patients. At four weeks ulcers had healed in 41/61 (67%) of patients taking the morning dose and in 47/63 (75%) of those receiving the nocturnal dose (95% CI for the difference -0.09 +0.25; p ns). At eight weeks, the corresponding healing rates were 82% and 85.5%, respectively (95% CI for the difference -0.11 +0.17; p ns). Both treatments were significantly superior to placebo in raising 24 h intragastric pH, although the effects of the morning dose were of shorter duration than those of the nocturnal dose. These findings suggest that suppression of nocturnal acidity is important but not essential to promote healing of duodenal ulcers; a prolonged period of acid inhibition during the day (as obtained with a single large morning dose of H2-blockers) may be equally effective. PMID:2186980

  14. Unaffected arm muscle hypercatabolism in dysphagic subacute stroke patients: the effects of essential amino acid supplementation.

    PubMed

    Aquilani, Roberto; Boselli, Mirella; D'Antona, Giuseppe; Baiardi, Paola; Boschi, Federica; Viglio, Simona; Iadarola, Paolo; Pasini, Evasio; Barbieri, Annalisa; Dossena, Maurizia; Bongiorno, Andria Innocenza; Verri, Manuela

    2014-01-01

    Alterations in muscle protein turnover of the unaffected side of stroke patients could contribute to physical disability. We investigated whether hypercatabolic activity occurred in unaffected arm muscle and whether supplemented essential amino acids (EAAs) could limit muscle hypercatabolism (MH). Thirty-eight dysphagic subacute stroke subjects (<3 months after acute event) (29 males+9 females; 69.7±11.4 yrs) were enrolled and randomized to receive 8 g/day EAAs (n=19; EAA group) or isocaloric placebo (maltodextrin; n=19, Plac group). Before randomization, all patients had their arterial (A) and venous (V) amino acids measured and muscle (A-V) differences calculated in the unaffected arm. Eight matched and healthy subjects served as controls. When compared to healthy controls, the entire stroke population showed significant muscle release (=negative value A-V) of the amino acid phenylalanine (phenyl-) indicating a prevalence of MH. Moreover, randomized EAA and Plac groups had similar rates of MH. After 38 days from the start of the protocol, the EAA group but not the Plac group had MH converted to balanced protein turnover or anabolic activity. We concluded that muscle protein metabolism of the unaffected arm of dysphagic subacute stroke individuals could be characterized by MH which can be corrected by supplemented EAAs. PMID:25431770

  15. Composition, texture and methane potential of cellulosic residues from Lewis acids organosolv pulping of wheat straw.

    PubMed

    Constant, Sandra; Barakat, Abdellatif; Robitzer, Mike; Di Renzo, Francesco; Dumas, Claire; Quignard, Françoise

    2016-09-01

    Cellulosic pulps have been successfully isolated from wheat straw through a Lewis acids organosolv treatment. The use of Lewis acids with different hardness produced pulps with different delignification degrees. The cellulosic residue was characterised by chemical composition, X-ray diffraction, FT-IR spectroscopy, N2 physisorption, scanning electron microscopy and potential for anaerobic digestibility. Surface area and pore volume increased with the hardness of the Lewis acid, in correspondence with the decrease of the amount of lignin and hemicellulose in the pulp. The non linearity of the correlation between porosity and composition suggests that an agglomeration of cellulose fibrils occurs in the early stages of pulping. All organosolv pulps presented a significantly higher methane potential than the parent straw. A methane evolution of 295Ncm(3)/g OM was reached by a moderate improvement of the accessibility of the native straw. PMID:27295251

  16. A Novel Treatment for Acid Mine Drainage Utilizing Reclaimed Limestone Residual

    SciTech Connect

    Horace K. Moo-Young; Charles E. Ochola

    2004-08-31

    The viability of utilizing Reclaimed Limestone Residual (RLR) to remediate Acid Mine Drainage (AMD) was investigated. Physical and chemical characterization of RLR showed that it is composed of various minerals that contain significant quantities of limestone or calcium bearing compounds that can be exploited for acid neutralization. Acid Neutralization Potential (ANP) test results showed that RLR has a neutralization potential of approximately 83% as calcium carbonate (CaCO{sub 3}). Neutralization tests with most of the heavy metals associated with AMD showed removal efficiencies of over 99%. An unexpected benefit of utilizing RLR was the removal of hexavalent chromium Cr (VI) from the aqueous phase. Due to an elevation in pH by RLR most AMD heavy metals are removed from solution by precipitation as their metal hydroxides. Cr (VI) however is not removed by pH elevation and therefore subsequent ongoing tests to elucidate the mechanism responsible for this reaction were conducted.

  17. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined. PMID:9230476

  18. Functional role of polar amino acid residues in Na+/H+ exchangers.

    PubMed Central

    Wiebe, C A; Dibattista, E R; Fliegel, L

    2001-01-01

    Na(+)/H(+) exchangers are a family of ubiquitous membrane proteins. In higher eukaryotes they regulate cytosolic pH by removing an intracellular H(+) in exchange for an extracellular Na(+). In yeast and Escherichia coli, Na(+)/H(+) exchangers function in the opposite direction to remove intracellular Na(+) in exchange for extracellular H(+). Na(+)/H(+) exchangers display an internal pH-sensitivity that varies with the different antiporter types. Only recently have investigations examined the amino acids involved in pH-sensitivity and in cation binding and transport. Histidine residues are good candidates for H(+)-sensing amino acids, since they can ionize within the physiological pH range. Histidine residues have been shown to be important in the function of the E. coli Na(+)/H(+) exchanger NhaA and in the yeast Na(+)/H(+) exchanger sod2. In E. coli, His(225) of NhaA may function to interact with, or regulate, the pH-sensory region of NhaA. In sod2, His(367) is also critical to transport and may be a functional analogue of His(225) of NhaA. Histidine residues are not critical for the function of the mammalian Na(+)/H(+) exchanger, although an unusual histidine-rich sequence of the C-terminal tail has some influence on activity. Other amino acids involved in cation binding and transport by Na(+)/H(+) exchangers are only beginning to be studied. Amino acids with polar side chains such as aspartate and glutamate have been implicated in transport activity of NhaA and sod2, but have not been studied in the mammalian Na(+)/H(+) exchanger. Further studies are needed to elucidate the mechanisms involved in pH-sensitivity and cation binding and transport by Na(+)/H(+) exchangers. PMID:11415429

  19. Particulates in hydrometallurgy: Part III. Dewatering behavior of flocculated laterite acid leach residues

    NASA Astrophysics Data System (ADS)

    Briceno, A.; Osseo-Asare, K.

    1995-02-01

    Three polyacrylamide-based polymers of different chemical properties (polymer A, 34 pct anionic, 11×106 mol wt; polymer B, 7 pct anionic, 7.5×106 mol wt; polymer C, nonionic, 13.5×106 mol wt) were used to evaluate the flocculation behavior of laterite acid leach residues. The solid-liquid separation characteristics of the leach residues were investigated with the aid of settling rate, supernatant turbidity, and slurry filtrability measurements. The polymeric flocculants were found to be effective in improving the dewatering properties of the acid leach residues. Polymer effectiveness increased with increasing polymer dosage for all the polymers, but an optimum polymer dose was only found for polymer A (34 pct anionic, 11×106 mol wt) in the studied range of polymer addition. Similarly, the dewatering behavior was improved at higher polymer molecular weight. In addition, it was found that the flocculation performance was adversely affected by an increase in the degree of polymer hydrolysis which, in turn, increases the ratio of carboxylic to amide functional groups in the polymer chain. Polymer C (nonionic ˜0 pct hydrolysis, 13.5×106 mol wt) was found to be the most efficient flocculant in terms of all the performance criteria investigated. The preceding results were rationalized in terms of bridging flocculation, the ionization and molecular configuration of the polymers, hydrogen bonding, and the solid/aqueous interfacial charge.

  20. Radionuclide Leaching from Residual Solids Remaining after Acid Dissolution of Composite K East Canister Sludge

    SciTech Connect

    Delegard, C.H.; Rinehart, D.E.; Soderquist, C.Z.; Fadeff, S.K.

    1999-04-02

    Laboratory tests were performed to examine mixed nitric/hydrofluoric acid leach treatments for decontaminating dissolver residual solids (KECDVSR24H-2) produced during a 20- to 24-hr dissolution of a composite K East (KE) Basin canister sludge in 95 C 6 M nitric acid (HNO{sub 3}). The scope of this testing has been described in Section 4.5 of ''Testing Strategy to Support the Development of K Basin Sludge Treatment Process'' (Flament 1998). Radionuclides sorbed or associated with the residual solids generated in the K Basin sludge treatment process can restrict disposal of this solid to the Environmental Restoration Disposal Facility (ERDF). The starting dissolver residual solid for this testing, KECDVSR24H-2, contains radionuclides at concentrations which exceed the ERDF Waste Acceptance Criteria for TRU by about a factor of 70, for {sup 239}Pu by a factor of 200, and for {sup 241}Am by a factor of 50. The solids also exceed the ERDF criterion for {sup 137}Cs by a factor of 2 and uranium by a factor of 5. Therefore, the radionuclides of greatest interest in this leaching study are first {sup 239}Pu and {sup 241}Am (both components of TRU) and then uranium and {sup 137}Cs.

  1. Degradation of articular cartilage keratan sulphates using hydrazinolysis and nitrous acid. Environment of fucose residues.

    PubMed Central

    Brown, G M; Huckerby, T N; Morris, H G; Nieduszynski, I A

    1992-01-01

    Alkaline borohydride-reduced keratan sulphate (KS) chains from bovine articular cartilage (6-8-year-old animals) were fragmented by an anhydrous hydrazine/nitrous acid procedure, previously used on KS by Hopwood & Elliott to isolate the major disaccharides from the poly-N-acetyl-lactosamine repeat sequence [Hopwood & Elliott (1983) Carbohydr. Res. 117, 263-274]. The resulting oligosaccharides were reduced with NaB3H4 or NaBH4 and subjected to ion-exchange chromatography on a Nucleosil 5SB column. In addition to the major disaccharides, two fucose-containing oligosaccharides were examined by high-field 1H n.m.r. spectroscopy, and shown to have the following structures (where AnManOH is 2,5-anhydro-D-mannitol): [formula: see text] It is evident that the presence of fucose protects the N-acetylglucosamine residue from de-N-acetylation, and therefore fragments are produced which preserve the immediate environment of the fucose residue. It may be of biosynthetic significance that these two oligosaccharides contain an unsulphated galactose on the non-reducing side of the fucose residue. The hydrazine/nitrous acid/NaB3H4 method followed by h.p.l.c. provides a sensitive fingerprinting technique for the assay of KS composition and sub-populations. PMID:1520275

  2. Methane emissions from beef cattle: effects of fumaric acid, essential oil, and canola oil.

    PubMed

    Beauchemin, K A; McGinn, S M

    2006-06-01

    The objective of this study was to identify feed additives that reduce enteric methane emissions from cattle. We measured methane emissions, total tract digestibility (using chromic oxide), and ruminal fermentation (4 h after feeding) in growing beef cattle fed a diet supplemented with various additives. The experiment was designed as a replicated 4 x 4 Latin square with 21-d periods and was conducted using 16 Angus heifers (initial BW of 260 +/- 32 kg). Treatments were: control (no additive), fumaric acid (175 g/d) with sodium bicarbonate (75 g/d), essential oil and spice extract (1 g/d), or canola oil (4.6% of DMI). The basal diet consisted of 75% whole-crop barley silage, 19% steam-rolled barley, and 6% supplement (DM basis). Four large chambers (2 animals fed the same diet per chamber) were equipped to measure methane emissions for 3 d each period. Adding canola oil to the diet decreased (P = 0.009) total daily methane emissions by 32% and tended (P = 0.09) to decrease methane emissions as a percentage of gross energy intake by 21%. However, much of the reduction in methane emissions was due to decreased (P < 0.05) feed intake and lower (P < 0.05) total tract digestibility of DM and fiber. Digestibility of all nutrients was also lowered (P < 0.05) by feeding essential oil, but there were no effects on ruminal fermentation or methane emissions. In contrast, adding fumaric acid to the diet increased total VFA concentration (P = 0.03), increased propionate proportions (P = 0.01), and decreased the acetate:propionate ratio (P = 0.002), but there was no measurable effect on methane emissions. The study demonstrates that canola oil can be used to reduce methane losses from cattle, but animal performance may be compromised due to lower feed intake and decreased fiber digestibility. Essential oils had no effect on methane emissions, whereas fumaric acid caused potentially beneficial changes in ruminal fermentation but no measurable reductions in methane emissions. PMID

  3. Leaching of lead from zinc leach residue in acidic calcium chloride aqueous solution

    NASA Astrophysics Data System (ADS)

    Wang, Le; Mu, Wen-ning; Shen, Hong-tao; Liu, Shao-ming; Zhai, Yu-chun

    2015-05-01

    A process with potentially reduced environmental impacts and occupational hazards of lead-bearing zinc plant residue was studied to achieve a higher recovery of lead via a cost-effective and environmentally friendly process. This paper describes an optimization study on the leaching of lead from zinc leach residue using acidic calcium chloride aqueous solution. Six main process conditions, i.e., the solution pH value, stirring rate, concentration of CaCl2 aqueous solution, liquid-to-solid (L/S) ratio, leaching temperature, and leaching time, were investigated. The microstructure and components of the residue and tailing were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). On the basis of experimental results, the optimum reaction conditions were determined to be a solution pH value of 1, a stirring rate of 500 r·min-1, a CaCl2 aqueous solution concentration of 400 g·L-1, a liquid-to-solid mass ratio of 7:1, a leaching temperature of 80°C, and a leaching time of 45 min. The leaching rate of lead under these conditions reached 93.79%, with an iron dissolution rate of 19.28%. Silica did not take part in the chemical reaction during the leaching process and was accumulated in the residue.

  4. Volatile fatty acids distribution during acidogenesis of algal residues with pH control.

    PubMed

    Li, Yan; Hua, Dongliang; Zhang, Jie; Zhao, Yuxiao; Xu, Haipeng; Liang, Xiaohui; Zhang, Xiaodong

    2013-06-01

    The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis. PMID:23381617

  5. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

    ...This notice announces the Agency's receipt of an initial filing of a pesticide petition proposing the establishment of a regulation for residues of the plant growth regulator, polymeric polyhydroxy acid, in or on all food...

  6. Essential amino acid transporter Lat4 (Slc43a2) is required for mouse development

    PubMed Central

    Guetg, Adriano; Mariotta, Luca; Bock, Lukas; Herzog, Brigitte; Fingerhut, Ralph; Camargo, Simone M R; Verrey, François

    2015-01-01

    Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development. Key points Lat4 (Slc43a2) transports branched-chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells. Using a new knockout model as a negative control, it is shown that Lat4 is expressed at the basolateral

  7. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  8. Determination of thymine glycol residues in irradiated or oxidized DNA by formation of methylglyceric acid

    SciTech Connect

    Schellenberg, K.A.; Shaeffer, J.

    1986-05-01

    Treatment of DNA solutions with X-irradiation various oxidants including hydrogen peroxide plus ferrous ion, hydrogen peroxide plus copper ion and ascorbate, permanganate, or sonication in the presence of dissolved oxygen all produced varying amounts of thymine glycol residues. After denaturing the DNA with heat, the glycol residues were reduced and labeled at the 6 position with tritium- labeled sodium borohydride. Subsequent reaction with anhydrous methanolic HCl gave a quantitative yield of the methyl ester of methylglyceric acid, which was determined by thin layer chromatography. The method, developed using thymidine as a model, was used to ascertain the requirements for glycol formation in DNA. It was shown that hydroxyl radical generating systems, permanganate, X-irradiation, or sonication in presence of oxygen were required, but hydrogen peroxide in the absence of iron or copper and ascorbate was inactive. Application to determination of DNA damage in vivo is being explored.

  9. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  10. Essential fatty acid profiling for routine nutritional assessment unmasks adrenoleukodystrophy in an infant with isovaleric acidaemia.

    PubMed

    Bonilla Guerrero, R; Wolfe, L A; Payne, N; Tortorelli, S; Matern, D; Rinaldo, P; Gavrilov, D; Melan, M; He, M; Steinberg, S J; Raymond, G V; Vockley, J; Gibson, K M

    2008-12-01

    We report a 16-month-old asymptomatic male with enzyme confirmed isovaleric acidaemia (IVA; isovaleryl-CoA dehydrogenase deficiency; OMIM 243500) who, upon routine nutritional follow-up, presented evidence of peroxisomal dysfunction. The newborn screen (2 days of life) revealed elevated C(5)-carnitine (2.95 μmol/L; cutoff <0.09 μmol/L) and IVA was subsequently confirmed by metabolic profiling and in vitro enzymology. Plasma essential fatty acid (EFA) analysis, assessed to evaluate nutritional status during protein restriction and L: -carnitine supplementation, revealed elevated C(26:0) (5.0 μmol/L; normal <1.3). Subsequently, metabolic profiling and molecular genetic analysis confirmed X-linked adrenoleukodystrophy (XALD). Identification of co-inherited XALD with IVA in this currently asymptomatic patient holds significant treatment ramifications for the proband prior to the onset of neurological sequelae, and critically important counselling implications for this family. PMID:19089597

  11. Symbiotic essential amino acids provisioning in the American cockroach, Periplaneta americana (Linnaeus) under various dietary conditions

    PubMed Central

    Larsen, Thomas; Sabree, Zakee

    2016-01-01

    Insect gut microbes have been shown to provide nutrients such as essential amino acids (EAAs) to their hosts. How this symbiotic nutrient provisioning tracks with the host’s demand is not well understood. In this study, we investigated microbial essential amino acid (EAA) provisioning in omnivorous American cockroaches (Periplaneta americana), fed low-quality (LQD) and comparatively higher-quality dog food (DF) diets using carbon stable isotope ratios of EAAs (δ13CEAA). We assessed non-dietary EAA input, quantified as isotopic offsets (Δ13C) between cockroach (δ13CCockroach EAA) and dietary (δ13CDietary EAA) EAAs, and subsequently determined biosynthetic origins of non-dietary EAAs in cockroaches using 13C-fingerprinting with dietary and representative bacterial and fungal δ13CEAA. Investigation of biosynthetic origins of de novo non-dietary EAAs indicated bacterial origins of EAA in cockroach appendage samples, and a mixture of fungal and bacterial EAA origins in gut filtrate samples for both LQD and DF-fed groups. We attribute the bacteria-derived EAAs in cockroach appendages to provisioning by the fat body residing obligate endosymbiont, Blattabacterium and gut-residing bacteria. The mixed signatures of gut filtrate samples are attributed to the presence of unassimilated dietary, as well as gut microbial (bacterial and fungal) EAAs. This study highlights the potential impacts of dietary quality on symbiotic EAA provisioning and the need for further studies investigating the interplay between host EAA demands, host dietary quality and symbiotic EAA provisioning in response to dietary sufficiency or deficiency. PMID:27231663

  12. Symbiotic essential amino acids provisioning in the American cockroach, Periplaneta americana (Linnaeus) under various dietary conditions.

    PubMed

    Ayayee, Paul A; Larsen, Thomas; Sabree, Zakee

    2016-01-01

    Insect gut microbes have been shown to provide nutrients such as essential amino acids (EAAs) to their hosts. How this symbiotic nutrient provisioning tracks with the host's demand is not well understood. In this study, we investigated microbial essential amino acid (EAA) provisioning in omnivorous American cockroaches (Periplaneta americana), fed low-quality (LQD) and comparatively higher-quality dog food (DF) diets using carbon stable isotope ratios of EAAs (δ (13)CEAA). We assessed non-dietary EAA input, quantified as isotopic offsets (Δ(13)C) between cockroach (δ (13)CCockroach EAA) and dietary (δ (13)CDietary EAA) EAAs, and subsequently determined biosynthetic origins of non-dietary EAAs in cockroaches using (13)C-fingerprinting with dietary and representative bacterial and fungal δ (13)CEAA. Investigation of biosynthetic origins of de novo non-dietary EAAs indicated bacterial origins of EAA in cockroach appendage samples, and a mixture of fungal and bacterial EAA origins in gut filtrate samples for both LQD and DF-fed groups. We attribute the bacteria-derived EAAs in cockroach appendages to provisioning by the fat body residing obligate endosymbiont, Blattabacterium and gut-residing bacteria. The mixed signatures of gut filtrate samples are attributed to the presence of unassimilated dietary, as well as gut microbial (bacterial and fungal) EAAs. This study highlights the potential impacts of dietary quality on symbiotic EAA provisioning and the need for further studies investigating the interplay between host EAA demands, host dietary quality and symbiotic EAA provisioning in response to dietary sufficiency or deficiency. PMID:27231663

  13. Bioaccumulation patterns of methyl mercury and essential fatty acids in lacustrine planktonic food webs and fish.

    PubMed

    Kainz, Martin; Telmer, Kevin; Mazumder, Asit

    2006-09-01

    Organisms of the planktonic food web convey essential nutrients as well as contaminants to animals at higher trophic levels. We measured concentrations of methyl mercury (MeHg) and essential fatty acids (EFAs, key nutrients for aquatic food webs) in four size categories of planktonic organisms - seston (10-64 microm), micro-(100-200 microm), meso-(200-500 microm), and macrozooplankton (>500 microm) - as well as total mercury (THg) and EFAs in rainbow trout (Oncorhynchus mykiss) in coastal lakes. We demonstrate that, in all lakes during this summer sampling, MeHg concentrations of planktonic organisms increase significantly with plankton size, independent of their taxonomic composition, and that their MeHg accumulation patterns predict significantly THg concentrations in rainbow trout (R2=0.71, p<0.05). However, concentrations of total EFAs do not follow this pattern. Total EFAs increased from seston to mesozooplankton but decreased in the largest zooplankton size fraction. Moreover, concentrations of individual EFA compounds in rainbow trout are consistently lower, with the exception of docosahexaenoic acid, than those in macrozooplankton. The continuous increase of MeHg concentrations in aquatic organisms, therefore, differs from patterns of EFA accumulation in zooplankton and fish. We interpret these contrasting accumulation patterns of MeHg and EFA compounds as the inability of aquatic organisms to regulate the assimilation of dietary MeHg, whereas the rate of EFA retention may be controlled to optimize their physiological performance. Therefore, we conclude that bioaccumulation patterns of Hg in these aquatic food webs are not controlled by lipid solubility and/or the retention of EFA compounds. PMID:16226794

  14. The importance of the ratio of omega-6/omega-3 essential fatty acids.

    PubMed

    Simopoulos, A P

    2002-10-01

    Several sources of information suggest that human beings evolved on a diet with a ratio of omega-6 to omega-3 essential fatty acids (EFA) of approximately 1 whereas in Western diets the ratio is 15/1-16.7/1. Western diets are deficient in omega-3 fatty acids, and have excessive amounts of omega-6 fatty acids compared with the diet on which human beings evolved and their genetic patterns were established. Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA) and a very high omega-6/omega-3 ratio, as is found in today's Western diets, promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a low omega-6/omega-3 ratio) exert suppressive effects. In the secondary prevention of cardiovascular disease, a ratio of 4/1 was associated with a 70% decrease in total mortality. A ratio of 2.5/1 reduced rectal cell proliferation in patients with colorectal cancer, whereas a ratio of 4/1 with the same amount of omega-3 PUFA had no effect. The lower omega-6/omega-3 ratio in women with breast cancer was associated with decreased risk. A ratio of 2-3/1 suppressed inflammation in patients with rheumatoid arthritis, and a ratio of 5/1 had a beneficial effect on patients with asthma, whereas a ratio of 10/1 had adverse consequences. These studies indicate that the optimal ratio may vary with the disease under consideration. This is consistent with the fact that chronic diseases are multigenic and multifactorial. Therefore, it is quite possible that the therapeutic dose of omega-3 fatty acids will depend on the degree of severity of disease resulting from the genetic predisposition. A lower ratio of omega-6/omega-3 fatty acids is more desirable in reducing the risk of many of the chronic diseases of high prevalence in Western societies, as well as in the developing countries, that are being exported to the rest of the world. PMID:12442909

  15. SAGA is an essential in vivo target of the yeast acidic activator Gal4p

    PubMed Central

    Bhaumik, Sukesh R.; Green, Michael R.

    2001-01-01

    Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood. For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets. Here we address these issues for the prototype acidic activator yeast Gal4p. Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose. Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA (Spt/Ada/GCN5/acetyltransferase) complex. Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS. If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur. SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites. Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5. Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription. PMID:11485988

  16. Toxicity of boric acid to Blattella germanica (Dictyoptera: Blattellidae) and analysis of residues in several organs.

    PubMed

    Habes, D; Kilani-Morakchi, S; Aribi, N; Farine, J P; Soltani, N

    2001-01-01

    Pestiferous cockroach species are associated closely with humans and are important from medical and public health points of view. Conventional insecticides have been used widely to control cockroaches which have developed resistance to these compounds. Thus, interest has again centered on lesser-used compounds such as boric acid. Boric acid has been used as an insecticide for many years, especially against cockroach. Its mode of action on insects has not been satisfactorily established. In Algeria, Blattella germanica (Dictyoptera: Blattellidae) is a serious pest in the urban environment and their infestation were controlled for many years by organophosphate, carbamate or pyrethroid insecticides. In order to obtain more information on the mode of action of boric acid, we first evaluated the oral toxicity of boric acid on B. germanica adults. Then, the compound was determined in several organs by an colorimetric method. This insecticide was incorporated into the diet and orally administered at different concentrations ranging from 1 to 40% (w/w) to newly emerged adults. Mortality was recorded at different times during treatment (24, 48, 72 and 144 h). Treatment resulted in a dose-dependent mortality since the LD50 (%) recorded are 85 at 24 h, 67 at 48 h, 39 at 72 h and 8 at 144 h, respectively. Then the quantity of boric acid accumulated in several organs (hemolymph, gut, ovaries, testicles and fat body) was determined as function the duration of treatment (1 to 5 days) for two doses (LD50 and LD90). Results revealed that bioaccumulation of residues in these organs increased as function the duration of treatment. In addition, relatively important amounts of residues, are detected in fat body. PMID:12425074

  17. Fatty acids, essential oil, and phenolics modifications of black cumin fruit under NaCl stress conditions.

    PubMed

    Bourgou, Soumaya; Bettaieb, Iness; Saidani, Moufida; Marzouk, Brahim

    2010-12-01

    This research evaluated the effect of saline conditions on fruit yield, fatty acids, and essential oils compositions and phenolics content of black cumin (Nigella sativa). This plant is one of the most commonly found aromatics in the Mediterranean kitchen. Increasing NaCl levels to 60 mM decreased significantly the fruits yield by 58% and the total fatty acids amount by 35%. Fatty acids composition analysis indicated that linoleic acid was the major fatty acid (58.09%) followed by oleic (19.21%) and palmitic (14.77%) acids. Salinity enhanced the linoleic acid percentage but did not affect the unsaturation degree of the fatty acids pool and thus the oil quality. The essential oil yield was 0.39% based on the dry weight and increased to 0.53, 0.56, and 0.72% at 20, 40, and 60 mM NaCl. Salinity results on the modification of the essential oil chemotype from p-cymene in controls to γ-terpinene/p-cymene in salt-stressed plants. The amounts of total phenolics were lower in the treated plants. Salinity decreased mainly the amount of the major class, benzoics acids, by 24, 29, and 44% at 20, 40, and 60 mM NaCl. The results suggest that salt treatment may regulate bioactive compounds production in black cumin fruits, influencing their nutritional and industrial values. PMID:21049998

  18. Effect of lime on the availability of residual phosphorus and its extractability by dilute acid

    SciTech Connect

    Rhue, R.D.; Hensel, D.R.

    1983-01-01

    The objective of this study was to determine the long-term effects of liming an acid, P-deficient Placid sand (sandy, siliceous, hyperthermic Typic Humaquept) on the availability of residual fertilizer P to potatoes (Solanum tuberosum L.). Dolomitic limestone was applied in November 1977, at rates of 0, 2240, 4480, and 8960 kg/ha in a split-plot design with lime as main plots and P treatments as subplots. Phosphorus was applied at rates of 0, 56, 112, and 168 kg/ha in 1978. In 1979 and 1980, P plots were split with one-half fertilized with 56 kg P/ha and the other one-half not fertilized with P (residual). In 1978, maximum tuber yields and top dry weights occurred at the 2240 kg/ha lime rate which resulted in a soil pH of 5.8. Plant P concentrations were unaffected by lime at any sampling rate. In 1979, availability of residual soil P decreased with lime rates > 2240 kg/ha but not enough to significantly affect yields. However, in 1980, overliming injury was observed for tuber yields at the higher lime rates which was the result of P deficiency. Application of P at planting eliminated the overliming injury with maximum yields occurring in the pH range of 6.0 to 6.5. It appears that liming to pH 6.5 in this study resulted in fertilizer reaction products that were more soluble in dilute acid but less plant available than those formed under more acid conditions. However, the Mehlich I extractant appeared to be a suitable extractant for P on this soil if pH was taken into account when interpreting soil-test P. 23 references, 4 figures, 2 tables.

  19. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by -rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

  20. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

    PubMed

    Addamiano, Claudia; Gerland, Béatrice; Payrastre, Corinne; Escudier, Jean-Marc

    2016-01-01

    Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. PMID:27563857

  1. Arginine becomes an essential amino acid after massive resection of rat small intestine.

    PubMed

    Wakabayashi, Y; Yamada, E; Yoshida, T; Takahashi, H

    1994-12-23

    We compared effects of feeding arginine- and/or proline- deficient diets (-Arg, -Pro, and -Arg, Pro) with those of a complete diet (Complete) in rats whose small intestine had been massively resected. After 4 weeks, the rats fed -Arg and -Arg, Pro lost weight (a mean of 28 and 32 g, respectively), whereas those fed Complete and -Pro gained 80 and 58 g, respectively. The average nitrogen balance was about 117,100, -20 and -14 mg/day for Complete, -Pro, -Arg, and -Arg, Pro diets, respectively. The concentration of arginine in skeletal muscle was about 310, 330, 91, and 65 nmol/g for Complete, -Pro, -Arg, and -Arg, Pro, respectively; while plasma arginine concentration averaged 95, 107, 56, and 46 microM, respectively. The weight loss, the negative nitrogen balance, and the markedly reduced arginine concentration in the muscle observed in rats fed -Arg and -Arg, Pro clearly indicate that arginine becomes a strictly essential amino acid in the rats with massive resection of the small intestine. However, sufficient proline can be synthesized from arginine in tissues such as the liver and kidney in the absence of the small intestine. Plasma glutamine, citrulline in the muscle and plasma, urinary excretion of orotic acid and nitrate (to assess nitric oxide formation from arginine) were also measured, and the changes in these metabolites are discussed. PMID:7798273

  2. Essential Fatty Acid Assimilation and Synthesis in Larvae of the Bivalve Crassostrea gigas.

    PubMed

    da Costa, Fiz; Robert, René; Quéré, Claudie; Wikfors, Gary H; Soudant, Philippe

    2015-05-01

    Essential fatty acids (EFA) are important for bivalve larval survival and growth. The purpose of this study was to quantitatively assess for the first time through a mass-balance approach dietary EFA incorporation and synthesis within Crassostrea gigas larvae. A first experiment was carried out using two microalgae, Tisochrysis lutea (T) and Chaetoceros neogracile (Cg), as mono- and bi-specific diets. A second experiment using a similar design was performed to confirm and extend the results obtained in the first. Flow-through larval rearing was used for accurate control of food supply and measurement of ingestion. Non-methylene-interrupted fatty acids were synthetized from precursors supplied in the diet: 16:1n-7 and 18:1n-9, mediated by Δ5 desaturase. Moreover, this Δ5 desaturase presumably allowed larvae to convert 20:3n-6 and 20:4n-3 to 20:4n-6 and 20:5n-3, respectively, when the product EFA were poorly or not supplied in the diet, as when larvae were fed T exclusively. Under our experimental conditions, none of the diets induced 22:6n-3 synthesis; however, 22:6n-3 incorporation into larval tissues occurred selectively under non-limiting dietary supply to maintain optimal levels in the larvae. This combination of flow-through larval rearing and biochemical analysis of FA levels could be applied to additional dietary experiments to precisely define optimal levels of EFA supply. PMID:25771891

  3. Comparative evaluation on fatty acid and Matricaria recutita essential oil incorporated into casein-based film.

    PubMed

    Aliheidari, Nahal; Fazaeli, Mahboubeh; Ahmadi, Reza; Ghasemlou, Mehran; Emam-Djomeh, Zahra

    2013-05-01

    Sodium caseinate composite films containing lipids-oleic acid (OA), stearic acid (SA), or Matricaria recutita essential oil (MEO) - were prepared through emulsification and their physical, thermal, mechanical, and barrier properties were evaluated and compared. Furthermore, their antimicrobial effectiveness against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli was studied. Emulsified films were softer, less rigid, and more stretchable than pure films. The films' water vapor barrier properties were found to decrease upon the addition of lipid content; this effect was greatly reduced when MEO was added. The presence of OA/SA and MEO decreased tensile strength and elastic modulus but increased the elongation at break. Thermal analysis of all emulsified films showed two endothermic peaks; these results confirmed those obtained by SEM studies, where a partial separation of the two phases occurred. The films' antimicrobial activities were increased by incorporating lipids, particularly those containing MEO, which were more effective against the studied bacteria. This work showed that when taking all the studied variables into account, films formulated with MEO were found most suitable for various food applications. PMID:23415659

  4. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

    PubMed

    Yang, Qing-Qing; Zhang, Chang-Quan; Chan, Man-Ling; Zhao, Dong-Sheng; Chen, Jin-Zhu; Wang, Qing; Li, Qian-Feng; Yu, Heng-Xiu; Gu, Ming-Hong; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2016-07-01

    Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice. PMID:27252467

  5. Fatty acids, essential oil and phenolics composition of Silybum marianum seeds and their antioxidant activities.

    PubMed

    Mhamdi, Baya; Abbassi, Feten; Smaoui, Abderrazak; Abdelly, Chedly; Marzouk, Brahim

    2016-05-01

    The presentstudydescribes the biochemical evaluation of Silybum marianum seed. The analysis of essential oil composition of Silybum marianum seed by Gas Chromatography-Mass Spectrometry GC-MS showed the presence of14 volatile components with the predominance of γ-cadinene (49.8%) and α-pinene (24.5%). Whereas, the analysis of fatty acids composition, showed the predominance of linoleic (50.5%) and oleic (30.2%) acids. Silybum marainum presented also an important polyphenol contents with 29mgGAE/g DW, a good antiradical activity (CI(50)=39μg/ml) but a lower reducing power ability. Flavonoid and condensed tannin contents were about 3.39mg EC/g DW and 1.8mg EC/gDW, respectively. The main phenolic compounds identified by RP-HPLC, were silybin A (12.2%), silybin B (17.67%), isosilybin A (21.9%), isosilybin B (12.8%), silychristin (7.9%) andsilydianin (7.5%). PMID:27166539

  6. Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

    PubMed

    Liou, H L; Storch, J

    2001-05-29

    The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions. PMID:11371211

  7. A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis.

    PubMed

    Guan, Xin; Chen, Hui; Abramson, Alex; Man, Huimin; Wu, Jinxia; Yu, Oliver; Nikolau, Basil J

    2015-11-01

    In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo-mtACP isoforms. The mitochondrial localization of the AT3G11470-encoded proteins was validated by the ability of their N-terminal 80-residue leader sequence to guide a chimeric GFP protein to this organelle. A T-DNA-tagged null mutant mtppt-1 allele shows an embryo-lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non-photorespiratory condition (i.e. 1% CO2 atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase. PMID:26402847

  8. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

    PubMed Central

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M.; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  9. Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

    PubMed

    Zhu, Feng; Xi, De-Hui; Yuan, Shu; Xu, Fei; Zhang, Da-Wei; Lin, Hong-Hui

    2014-06-01

    Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV. PMID:24450774

  10. Improving the acidic stability of Staphylococcus aureus α-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues.

    PubMed

    Zhang, Xian; Rao, Zhiming; Li, Jingjing; Zhou, Junping; Yang, Taowei; Xu, Meijuan; Bao, Teng; Zhao, Xiaojing

    2015-04-01

    The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K m and V max were 17.7 μM and 2.06 mM min(-1), respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL(-1) of SaALDC activity, about 320 times higher comparing to 0.42 U mL(-1) of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host. PMID:25543264

  11. Fractionation of the stable carbon isotope ratio of essential fatty acids in zebrafish Danio rerio and mud snails Bellamya chinensis.

    PubMed

    Fujibayashi, Megumu; Ogino, Masahiro; Nishimura, Osamu

    2016-02-01

    Fractionation of stable carbon (C) isotopes in the essential fatty acids 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3, and 22:6n-3 was investigated in the zebrafish Danio rerio and the mud snail Bellamya chinensis fed the same two diets. These diets differed in essential fatty acid compositions: (1) TetraMin contained all five fatty acids, and (2) Chlorella contained only two, 18:2n-6 and 18:3n-3. On average, the isotopic fractionation was -0.5 ± 0.9 ‰ for 18:2n-6 and 18:3n-3 for all experiments, indicating that the fractionation of these essential fatty acids was negligible. However, the isotopic fractionation of 20:4n-6, 20:5n-3, and 22:6n-3 varied greatly between species and between diets. The isotopic fractionation of the Chlorella diet was -0.2 and -6.9 ‰ for zebrafish and mud snail, but 4.2 and -1.3 ‰, respectively, when these consumers were fed TetraMin. This variation could be explained by the different amount of assimilation and the biosynthesis of these fatty acids from their precursors (i.e., 18:2n-6 and 18:3n-3). These results indicate that the isotopic composition of C20 and C22 essential fatty acids was strongly influenced by the fatty acid composition in the diets. Thus the stable C isotope ratios of C18 essential fatty acids in consumers are more useful as dietary tracers in food web studies. PMID:26537876

  12. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  13. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  14. Quantitative solid state NMR analysis of residues from acid hydrolysis of loblolly pine wood.

    PubMed

    Sievers, Carsten; Marzialetti, Teresita; Hoskins, Travis J C; Valenzuela Olarte, Mariefel B; Agrawal, Pradeep K; Jones, Christopher W

    2009-10-01

    The composition of solid residues from hydrolysis reactions of loblolly pine wood with dilute mineral acids is analyzed by (13)C Cross Polarization Magic Angle Spinning (CP MAS) NMR spectroscopy. Using this method, the carbohydrate and lignin fractions are quantified in less than 3h as compared to over a day using wet chemical methods. In addition to the quantitative information, (13)C CP MAS NMR spectroscopy provides information on the formation of additional extractives and pseudo lignin from the carbohydrates. Being a non-destructive technique, NMR spectroscopy provides unambiguous evidence of the presence of side reactions and products, which is a clear advantage over the wet chemical analytical methods. Quantitative results from NMR spectroscopy and proximate analysis are compared for the residues from hydrolysis of loblolly pine wood under 13 different conditions; samples were treated either at 150 degrees C or 200 degrees C in the presence of various acids (HCl, H(2)SO(4), H(3)PO(4), HNO(3) and TFA) or water. The lignin content determined by both methods differed on averaged by 2.9 wt% resulting in a standard deviation of 3.5 wt%. It is shown that solid degradation products are formed from saccharide precursors under harsh reaction conditions. These degradation reactions limit the total possible yield of monosaccharides from any subsequent reaction. PMID:19477123

  15. Resistance of essential fatty acid-deficient rats to endotoxin-induced increases in vascular permeability

    SciTech Connect

    Li, E.J.; Cook, J.A.; Spicer, K.M.; Wise, W.C.; Rokach, J.; Halushka, P.V. )

    1990-06-01

    Resistance to endotoxin in essential fatty acid-deficient (EFAD) rats is associated with reduced synthesis of certain arachidonic acid metabolites. It was hypothesized that EFAD rats would manifest decreased vascular permeability changes during endotoxemia as a consequence of reduced arachidonic acid metabolism. To test this hypothesis, changes in hematocrit (HCT) and mesenteric localization rate of technetium-labeled human serum albumin (99mTc-HSA) and red blood cells (99mTc-RBC) were assessed in EFAD and normal rats using gamma-camera imaging. Thirty minutes after Salmonella enteritidis endotoxin, EFAD rats exhibited less hemoconcentration as determined by % HCT than normal rats. Endotoxin caused a less severe change in permeability index in the splanchnic region in EFAD rats than in normal rats (1.2 +/- 0.6 x 10(-3)min-1 vs. 4.9 +/- 1.7 x 10(-3)min-1 respectively, P less than 0.05). In contrast to 99mTc-HSA, mesenteric localization of 99mTc-RBC was not changed by endotoxin in control or EFAD rats. Supplementation with ethyl-arachidonic acid did not enhance susceptibility of EFAD rats to endotoxin-induced splanchnic permeability to 99mTc-HSA. Leukotrienes have been implicated as mediators of increased vascular permeability in endotoxin shock. Since LTC3 formation has been reported to be increased in EFA deficiency, we hypothesized that LTC3 may be less potent than LTC4. Thus the effect of LTC3 on mean arterial pressure and permeability was compared to LTC4 in normal rats. LTC3-induced increases in peak mean arterial pressure were less than LTC4 at 10 micrograms/kg (39 +/- 5 mm Hg vs. 58 +/- 4 mm Hg respectively, P less than 0.05) and at 20 micrograms/kg (56 +/- 4 mm Hg vs. 75 +/- 2 mm Hg respectively, P less than 0.05). LY171883 (30 mg/kg), an LTD4/E4 receptor antagonist, attenuated the pressor effect of LTC4, LTD4, and LTC3.

  16. Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway*

    PubMed Central

    Pélissier, Marie-Cécile; Sebban-Kreuzer, Corinne; Guerlesquin, Françoise; Brannigan, James A.; Bourne, Yves; Vincent, Florence

    2014-01-01

    Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys66 as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. 1H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys66 for the epimerization reaction with participation of neighboring Arg43, Asp126, and Glu180 residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases. PMID:25320079

  17. The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

    PubMed Central

    Dagil, Yulia A.; Arbatsky, Nikolai P.; Alkhazova, Biana I.; L’vov, Vyacheslav L.; Mazurov, Dmitriy V.; Pashenkov, Mikhail V.

    2016-01-01

    Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants. PMID:27513337

  18. Kinetics of sulfuric acid leaching of cadmium from Cd-Ni zinc plant residues.

    PubMed

    Safarzadeh, Mohammad Sadegh; Moradkhani, Davood; Ojaghi-Ilkhchi, Mehdi

    2009-04-30

    Cd-Ni filtercakes are produced continuously at the third purification step in the electrolytic production of zinc in the National Iranian Lead and Zinc Company (NILZ) in northwestern Iran. In this research, the dissolution kinetics of cadmium from Cd-Ni residues produced in NILZ plant has been investigated. Hence, the effects of temperature, sulfuric acid concentration, particle size and stirring speed on the kinetics of cadmium dissolution in sulfuric acid were studied. The dissolution kinetics at 25-55 degrees C and tacid concentration, solid/liquid ratio and particle size were also achieved. The rate of reaction at first 5 min based on diffusion-controlled process can be expressed by a semi-empirical equation as:It was determined that the dissolution rate increased with increasing sulfuric acid concentration and decreasing particle size. PMID:18755541

  19. Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin.

    PubMed Central

    Watanabe, Takeshi; Ariga, Yumiko; Sato, Urara; Toratani, Tadayuki; Hashimoto, Masayuki; Nikaidou, Naoki; Kezuka, Yuichiro; Nonaka, Takamasa; Sugiyama, Junji

    2003-01-01

    Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure. PMID:12930197

  20. Effect of sex and gonadal hormones on rat plasma lipids during the development of an essential fatty acid deficiency

    PubMed Central

    Lyman, R. L.; Ostwald, Rosemarie; Bouchard, Pauline; Shannon, Angela

    1966-01-01

    1. Male, female and castrated rats treated with oestradiol (30μg./week) or testosterone (2mg./week) were given an essential fatty acid-deficient diet containing 10% of hydrogenated coconut oil for 9 weeks. The concentrations and fatty acid composition of plasma phospholipids, cholesteryl esters and triglycerides were determined. 2. Between the second and third weeks of the deficiency, concentrations of plasma cholesteryl esters, phospholipids and triglycerides decreased, then remained relatively constant. There were no significant differences between males and females, but oestradiol caused a significant rise in plasma phospholipids and triglycerides as compared with testosterone-treated animals. 3. During the first 2 weeks of the deficiency, linoleic acid in the plasma lipids of all groups decreased to low concentrations and changed very little thereafter. 4. Female rats maintained higher percentages and concentrations of arachidonic acid and stearic acid in plasma phospholipids and arachidonic acid in cholesteryl esters than did males. Males had higher proportions of eicosatrienoic acid and oleic acid. There was no sex difference in the fatty acid composition of plasma triglycerides. 5. Oestradiol-treated rats had concentrations of cholesteryl and phospholipid arachidonate comparable with those of female rats and higher than the testosterone-treated group. Eicosatrienoic acid in the oestradiol–treated rats was high and resembled that of the male rats, apparently because of the higher concentration of plasma phospho lipids in this group. 6. Supplementation of the essential fatty acid-deficient rats with linoleate restored plasma cholesteryl and phospholipid linoleate and arachidonate nearly to normal concentrations in a single day. The increase in arachidonic acid in these fractions was accompanied by a similar quantitative decrease in eicosatrienoic acid. 7. These sex differences appear to be related to the smaller size of the female rat and to a more direct

  1. Monomethyl Branched-Chain Fatty Acids Play an Essential Role in Caenorhabditis elegans Development

    PubMed Central

    Crawford, Quinn T; Seiber, Matt; Wang, Cun-Yu

    2004-01-01

    Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes. PMID:15340492

  2. Inhibition of the ATPase from Halobacterium Saccharovorum by Thiol Inhibitors: Evidence for the Presence of More Than One Essential Cysteinyl Residue

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Emrich, Errol; Stan-Lotter, Helga; DeVincenzi, Donald L. (Technical Monitor)

    1995-01-01

    The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

  3. Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

    PubMed

    Maravić, Gordana; Feder, Marcin; Pongor, Sándor; Flögel, Mirna; Bujnicki, Janusz M

    2003-09-01

    Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family. PMID:12946350

  4. Oral essential amino acid supplements in children with advanced chronic renal failure.

    PubMed

    Jones, R W; Dalton, N; Start, K; El-Bishti, M M; Chantler, C

    1980-07-01

    The effects on growth, nitrogen balance, and body composition of a protein-restricted diet supplemented with oral essential amino acids (EAA) were studied in seven children with advanced chronic renal failure. The diet was designed to provide minimum protein requirements for height-age, half in unselected form and half as an EAA supplement. Energy from carbohydrate and fat were increased to give a protein/energy ratio of 1.25 G:100 kcal. Nitrogen balance, studied in five children before and after 6 to 8 months of EAA treatment, was improved in each case. intracellular water (total body water minus bromide space) increased in four children but fell in three children during treatment. No significant improvement in growth, expressed as height or height velocity standard deviation scores in relation to bone age, was observed. Serum urea and urea/creatinine ratio fell after institution of EAA treatment, but the fall was not sustained. Although the EAA preparation proved acceptable to the children, dietary assessments indicated that the desired dietary aims were rarely achieved. It is concluded that, in this pediatric age group, the long-term application of a protein restricted diet with EAA supplements is of limited value. PMID:7395791

  5. Effect of essential fatty acid deficiency on experimental allergic encephalomyelitis in rats.

    PubMed

    Levine, S; Sowinski, R

    1980-05-01

    Previous claims that experimental allergic encephalomyelitis (EAE) was enhanced by essential fatty acid (EFA) deficiency were reinvestigated. Deficiency was induced in Lewis rats by feeding a fat-free diet starting in late gestation, at weaning or in adult life with or without a previous period of starvation. Retardation of growth, the typical dermatitis, increased water consumption and testicular atrophy gave evidence of EFA deficiency. Control rats were fed a complete diet or a fat-free diet supplemented with corn oil. EAE was induced in EFA-deficient and control rats by conventional active sensitization with neural antigen and adjuvants or by passive transfer of living lymphoid cells from sensitized nutritionally normal donors. Contrary to previous reports, EFA deficiency did not enhance EAE in any of seven experiments, and these results were supported by histological examinations. In fact, we found inhibition of clinical signs, but not histological lesions, when EFA deficiency was moderately advanced. This was accompanied by (and probably related to) thymic atrophy, possibly due to nonspecific stress. Also we found that EFA deficiency had no effect on a non-immunological model of brain inflammation that resembles EAE in the occurrence of lymphocytic infiltrates. PMID:7373435

  6. Hypercortisolemia alters muscle protein anabolism following ingestion of essential amino acids

    NASA Technical Reports Server (NTRS)

    Paddon-Jones, Douglas; Sheffield-Moore, Melinda; Creson, Daniel L.; Sanford, Arthur P.; Wolf, Steven E.; Wolfe, Robert R.; Ferrando, Arny A.

    2003-01-01

    Debilitating injury is accompanied by hypercortisolemia, muscle wasting, and disruption of the normal anabolic response to food. We sought to determine whether acute hypercortisolemia alters muscle protein metabolism following ingestion of a potent anabolic stimulus: essential amino acids (EAA). A 27-h infusion (80 microg. kg(-1). h(-1)) of hydrocortisone sodium succinate mimicked cortisol (C) levels accompanying severe injury (>30 microg/dl), (C + AA; n = 6). The control group (AA) received intravenous saline (n = 6). Femoral arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol. kg(-1). min(-1)) of l-[ring-(2)H(5)]phenylalanine before and after ingestion of 15 g of EAA. Hypercortisolemia [36.5 +/- 2.1 (C + AA) vs. 9.0 +/- 1.0 microg/dl (AA)] increased postabsorptive arterial, venous, and muscle intracellular phenylalanine concentrations. Hypercortisolemia also increased postabsorptive and post-EAA insulin concentrations. Net protein balance was blunted (40% lower) following EAA ingestion but remained positive for a greater period of time (60 vs. 180 min) in the C + AA group. Thus, although differences in protein metabolism were evident, EAA ingestion improved muscle protein anabolism during acute hypercortisolemia and may help minimize muscle loss following debilitating injury.

  7. Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S.

    PubMed

    Gouldson, P; Legoux, P; Carillon, C; Dumont, X; Le Fur, G; Ferrara, P; Shire, D

    2000-02-18

    We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor. PMID:10688974

  8. Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

    NASA Astrophysics Data System (ADS)

    Farias, Sávio Torres De; Moreira, Carlos Henrique Costa; Guimarães, Romeu Cardoso

    2007-02-01

    The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets N RR and N YY), distinguished from the mixed sector (triplets N RY and N YR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, C CC: G GG and A GA: U CU. This suggests that U CU previously belonged to Ser, adding to other indications that the attribution of Arg to Y CU was due to an expansion of the Arg- tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.

  9. Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

    PubMed Central

    2015-01-01

    The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217–Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed. PMID:25545070

  10. Intra-molecular cross-linking of acidic residues for protein structure studies.

    SciTech Connect

    Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr; Schoeniger, Joseph S.

    2005-03-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information

  11. Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

    PubMed

    Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

    2015-11-01

    Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. PMID:26196441

  12. Predicting the mobility of Zn, Fe, Cu, Pb, Cd from roasted sulfide (pyrite) residues -- A case study of wastes from the sulfuric acid industry in Sweden

    SciTech Connect

    Lin, Z.; Qvarfort, U.

    1996-12-31

    Leachates from roasted sulfide residues, which are the wastes from the production of sulfuric acid at Falun, Sweden, have low pH and high concentrations of Zn, Fe, and SO{sub 4}. The minerals are mainly hematite and maghemite and, because the various sulfides in the feed behave differently during the roasting process, the residual sulfides minerals are pyrrhotite and sphalerite. Oxidation of the residual sulfides contributes acidity, Zn, Fe, Cu, Cd, and sulfate to the effluents from the waste deposits. The dissolution of sphalerite is most likely accelerated in acid solution rich in Fe(III). The formation of Pb-sulfate coatings on galena may provide an armoring effect which slows the oxidation of the galena. Residual sulfides are source phases controlling long-term contaminant release. Other source minerals for Zn, Fe, Pb, Cu, Cd and SO{sub 4} in the effluents are iron oxides which retained percentage quantities of SO{sub 4}, roast-derived alteration rims of Zn oxides on sphalerite, altered silicates formed during the roasting process, and secondary minerals (e.g., Zn, Fe, Cu sulfates, iron hydroxides) that were precipitated by in-site oxidation in the waste dumps. The Zn, Fe, and Cu sulfates most likely control short-term changes in the chemistry of the leachate, while Pb concentration in the leachates may be controlled predominantly by Pb-release from the altered silicates. The mineralogical and geochemical data provide fundamental information essential for the remedial management of this type of industrial waste.

  13. Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

    PubMed Central

    2009-01-01

    Background Antibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown. Methods and Results In this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule. Conclusion In sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1. PMID:19751520

  14. Casein synthesis is independently and additively related to individual essential amino acid supply.

    PubMed

    Arriola Apelo, S I; Singer, L M; Ray, W K; Helm, R F; Lin, X Y; McGilliard, M L; St-Pierre, N R; Hanigan, M D

    2014-05-01

    Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h

  15. Leucine-enriched essential amino acids attenuate inflammation in rat muscle and enhance muscle repair after eccentric contraction.

    PubMed

    Kato, Hiroyuki; Miura, Kyoko; Nakano, Sayako; Suzuki, Katsuya; Bannai, Makoto; Inoue, Yoshiko

    2016-09-01

    Eccentric exercise results in prolonged muscle damage that may lead to muscle dysfunction. Although inflammation is essential to recover from muscle damage, excessive inflammation may also induce secondary damage, and should thus be suppressed. In this study, we investigated the effect of leucine-enriched essential amino acids on muscle inflammation and recovery after eccentric contraction. These amino acids are known to stimulate muscle protein synthesis via mammalian target of rapamycin (mTOR), which, is also considered to alleviate inflammation. Five sets of 10 eccentric contractions were induced by electrical stimulation in the tibialis anterior muscle of male SpragueDawley rats (8-9 weeks old) under anesthesia. Animals received a 1 g/kg dose of a mixture containing 40 % leucine and 60 % other essential amino acids or distilled water once a day throughout the experiment. Muscle dysfunction was assessed based on isometric dorsiflexion torque, while inflammation was evaluated by histochemistry. Gene expression of inflammatory cytokines and myogenic regulatory factors was also measured. We found that leucine-enriched essential amino acids restored full muscle function within 14 days, at which point rats treated with distilled water had not fully recovered. Indeed, muscle function was stronger 3 days after eccentric contraction in rats treated with amino acids than in those treated with distilled water. The amino acid mix also alleviated expression of interleukin-6 and impeded infiltration of inflammatory cells into muscle, but did not suppress expression of myogenic regulatory factors. These results suggest that leucine-enriched amino acids accelerate recovery from muscle damage by preventing excessive inflammation. PMID:27168073

  16. Characterisation of the products from pyrolysis of residues after acid hydrolysis of Miscanthus.

    PubMed

    Melligan, F; Dussan, K; Auccaise, R; Novotny, E H; Leahy, J J; Hayes, M H B; Kwapinski, W

    2012-03-01

    Platform chemicals such as furfural and hydroxymethylfurfural are major products formed during the acid hydrolysis of lignocellulosic biomass in second generation biorefining processes. Solid hydrolysis residues (HR) can amount to 50 wt.% of the starting biomass materials. Pyrolysis of the HRs gives rise to biochar, bio-liquids, and gases. Time and temperature were variables during the pyrolysis of HRs in a fixed bed tubular reactor, and both parameters have major influences on the amounts and properties of the products. Biochar, with potential for carbon sequestration and soil conditioning, composed about half of the HR pyrolysis product. The amounts (11-20 wt.%) and compositions (up to 77% of phenols in organic fraction) of the bio-liquids formed suggest that these have little value as fuels, but could be sources of phenols, and the gas can have application as a fuel. PMID:22281143

  17. Combined application of origanum vulgare l. essential oil and acetic acid for controlling the growth of staphylococcus aureus in foods

    PubMed Central

    de Souza, Evandro Leite; de Barros, Jefferson Carneiro; da Conceição, Maria Lúcia; Neto, Nelson Justino Gomes; da Costa, Ana Caroliny Vieira

    2009-01-01

    This study evaluated the occurrence of an enhancing inhibitory effect of the combined application of Origanum vulgare L. essential oil and acetic acid against Staphylococcus aureus by the determination of Fractional Inhibitory Concentration (FIC) index and kill-time assay in nutrient broth, meat broth and in a food model (meat pieces). Acetic acid showed MIC and MFC of 0.6 and 1.25 μL.mL-1, respectively. For O. vulgare essential oil MIC and MBC were 1.25 and 2.5 μL.mL-1, respectively. FIC indexes of the mixture of essential oil and acetic acid at MIC x ½ were ≤ 1.0, showing an additive effect. No synergy was found at kill-time study. Anti-staphylococcal effect of the antimicrobials alone or in mixture (MIC x ½) was lower in meat than in nutrient and meat broths. The effective combination of essential oils and organic acids could appear as an attractive alternative for the food industry, as the doses to inhibit the microbial growth in foods can be lowered. PMID:24031377

  18. Leucine-enriched essential amino acid supplementation during moderate steady state exercise enhances postexercise muscle protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The effects of essential amino acid (EAA) supplementation during moderate steady state (ie, endurance) exercise on postexercise skeletal muscle metabolism are not well described, and the potential role of supplemental leucine on muscle protein synthesis (MPS) and associated molecular re...

  19. A Mutational Analysis of Active Site Residues in trans-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Poelarends, Gerrit J.; Serrano, Hector; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    trans -3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations. PMID:23851010

  20. Selective conversion of cellulose in corncob residue to levulinic acid in an aluminum trichloride-sodium chloride system.

    PubMed

    Li, Jianmei; Jiang, Zhicheng; Hu, Libin; Hu, Changwei

    2014-09-01

    Increased energy consumption and environmental concerns have driven efforts to produce chemicals from renewable biomass with high selectivity. Here, the selective conversion of cellulose in corncob residue, a process waste from the production of xylose, to levulinic acid was carried out using AlCl3 as catalyst and NaCl as promoter by a hydrothermal method at relatively low temperature. A levulinic acid yield of 46.8 mol% was obtained, and the total selectivity to levulinic acid with formic acid was beyond 90%. NaCl selectively promoted the dissolution of cellulose from corncob residue, and significantly improved the yield and selectivity to levulinic acid by inhibiting lactic acid formation in the subsequent dehydration process. Owing to the salt effect of NaCl, the obtained levulinic acid could be efficiently extracted to tetrahydrofuran from aqueous solution. The aqueous solution with AlCl3 and NaCl could be recycled 4 times. Because of the limited conversion of lignin, this process allows for the production of levulinic acid with high selectivity directly from corncob residue in a simple separation process. PMID:25045141

  1. Residue Geometry Networks: A Rigidity-Based Approach to the Amino Acid Network and Evolutionary Rate Analysis.

    PubMed

    Fokas, Alexander S; Cole, Daniel J; Ahnert, Sebastian E; Chin, Alex W

    2016-01-01

    Amino acid networks (AANs) abstract the protein structure by recording the amino acid contacts and can provide insight into protein function. Herein, we describe a novel AAN construction technique that employs the rigidity analysis tool, FIRST, to build the AAN, which we refer to as the residue geometry network (RGN). We show that this new construction can be combined with network theory methods to include the effects of allowed conformal motions and local chemical environments. Importantly, this is done without costly molecular dynamics simulations required by other AAN-related methods, which allows us to analyse large proteins and/or data sets. We have calculated the centrality of the residues belonging to 795 proteins. The results display a strong, negative correlation between residue centrality and the evolutionary rate. Furthermore, among residues with high closeness, those with low degree were particularly strongly conserved. Random walk simulations using the RGN were also successful in identifying allosteric residues in proteins involved in GPCR signalling. The dynamic function of these residues largely remain hidden in the traditional distance-cutoff construction technique. Despite being constructed from only the crystal structure, the results in this paper suggests that the RGN can identify residues that fulfil a dynamical function. PMID:27623708

  2. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  3. Radiogenic Ar retention in residual silica from acid-treated micas

    NASA Astrophysics Data System (ADS)

    Derkowski, Arkadiusz; Szczerba, Marek; Środoń, Jan; Banaś, Michał

    2014-03-01

    In sedimentary basins, immediate equilibration with surface and pore waters of Ar, released from K-bearing minerals during their diagenesis or weathering, has been a paradigm for geochemistry and geochronology. Consequently, K-Ar and Ar-Ar isotope geochronology techniques applied to sedimentary rocks are based on an assumption that no measurable external radiogenic 40Ar (“excess argon”) has been locked in the rock components during their formation and alteration. Our results indicate that the reaction of micaceous sedimentary and diagenetic clay minerals (illite, glauconite) with acid produces microporous silica that retains a great fraction of the initial argon, releasing potassium to the solution. In all tested cases the evolution of K-Ar isotope ages followed the very same pattern: the apparent K-Ar isotope age increased enormously after acid treatment and dropped significantly after silica removal (with hot Na2CO3), but never decreased lower than the initial K-Ar isotope age of the untreated sample. The amorphous silica content and the apparent K-Ar age increased with the acid reaction time. Using the molecular dynamics simulations, the clay-acid reaction by-product was shown to bend and wrap, producing three-dimensional, protonated and hydrated silica. As a consequence of dramatically different hydration energies of Ar and K, potassium is instantaneously released and hydrated outside the residual structure while Ar atoms remain inside the silica network, adsorbed on the surface. This is, to our knowledge, the first experimental evidence that the excess argon can be retained in solid mineral reaction products formed under pressure and temperature close to those of the Earth surface (1 atm, <80 °C).

  4. Sonic hedgehog signaling directly targets Hyaluronic Acid Synthase 2, an essential regulator of phalangeal joint patterning.

    PubMed

    Liu, Jiang; Li, Qiang; Kuehn, Michael R; Litingtung, Ying; Vokes, Steven A; Chiang, Chin

    2013-03-15

    Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis. PMID:23313125

  5. Dose-escalation study of octanoic acid in patients with essential tremor

    PubMed Central

    Voller, Bernhard; Lines, Emily; McCrossin, Gayle; Tinaz, Sule; Lungu, Codrin; Grimes, George; Starling, Judith; Potti, Gopal; Haubenberger, Dietrich

    2016-01-01

    BACKGROUND. Recently, 1-octanol has been shown to have efficacy in treating patients with essential tremor (ET). The primary metabolite of 1-octanol is octanoic acid (OA), which is now thought to be the active substance that mediates tremor suppression. Our aim was to describe the maximum tolerated dose (MTD) of oral OA in patients with ET and assess the pharmacokinetics (PK) and pharmacodynamics (PD) profile of OA. METHODS. The MTD was studied using an open-label, single-ascending 3 + 3 dose–escalation design. Predefined single doses ranged from 8 to 128 mg/kg, with grade 2 adverse events (AEs) defined as dose-limiting toxicity. Tremor was assessed using accelerometry, digital spiral analysis, and a standard clinical rating scale at baseline and up to 600 minutes after intake. Safety assessments and PK sampling were also performed. RESULTS. Dose-limiting toxicity was not reached. The most frequent AE was mild abdominal discomfort. Exposure (AUC) increased linearly with the dose. Secondary efficacy measures suggested a dose-dependent reduction of tremor. Accordingly, a single unified PK/PD model with an effect compartment and sigmoid maximum effect (Emax) response could be built that accounted well for the time profiles of plasma concentrations as well as effects on tremor severity across the 5 dose levels. CONCLUSION. Although our trial did not reach an MTD, a dose-dependent effect was demonstrated in the PK/PD model as well as in secondary efficacy outcomes. Future studies are needed to explore the safety in higher dose ranges and to confirm dose-dependent efficacy in a placebo-controlled design. TRIAL REGISTRATION. Clinicaltrials.gov NCT01468948 FUNDING. NINDS Intramural Research Program; TG Therapeutics Inc. PMID:26927672

  6. Turnover of mitochondrial components of normal and essential fatty acid-deficient rats

    PubMed Central

    Bailey, E.; Taylor, C. B.; Bartley, W.

    1967-01-01

    1. Essential fatty acid (EFA)-deficient and control rats were injected intraperitoneally with [32P]phosphate, l-[35S]methionine and [2-14C]acetate. The animals were killed at various time-intervals after injection and their liver mitochondria fractionated into soluble protein, insoluble protein, and lipid. 2. The 35S was assayed in the protein fractions and 32P and 14C were assayed in the lipid fraction. Curves of log (specific activity) plotted against time were prepared for the different fractions. 3. There was no significant difference between the insoluble protein results for control and EFA-deficient animals, both sets of results indicating the presence of a single component of half-life 9 days. 4. There was no significant difference between the soluble protein results for the two sets of animals and both sets of results indicated the presence of at least two components. 5. The [32P]-phospholipid results indicate that in the control animals the liver mitochondrial phospholipids contain components of half-life 1·6 and 10 days whereas the mitochondrial phospholipids of the EFA-deficient animals contain components of half-life 3 and 29 days. 6. The specific activity of mitochondrial [14C]phospholipid initially fell rapidly in both groups of animals, but after 17 days there was no further significant decrease. A fast component with maximum half-life 2–4 days was clearly demonstrated for both groups of animals. Whether or not these results also indicate the presence of a very long-lived mitochondrial phospholipid is discussed. PMID:6049854

  7. Essential Amino Acid Supplementation by Gut Microbes of a Wood-Feeding Cerambycid.

    PubMed

    Ayayee, Paul A; Larsen, Thomas; Rosa, Cristina; Felton, Gary W; Ferry, James G; Hoover, Kelli

    2016-02-01

    Insects are unable to synthesize essential amino acids (EAAs) de novo, thus rely on dietary or symbiotic sources for them. Wood is a poor resource of nitrogen in general, and EAAs in particular. In this study, we investigated whether gut microbiota of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), a cerambycid that feeds in the heartwood of healthy host trees, serve as sources of EAAs to their host under different dietary conditions. δ(13)C-stable isotope analyses revealed significant δ(13)C-enrichment (3.4 ± 0.1‰; mean ± SEM) across five EAAs in wood-fed larvae relative to their woody diet. δ(13)C values for the consumers greater than 1‰ indicate significant contributions from non-dietary EAA sources (symbionts in this case). In contrast, δ(13)C-enrichment of artificial diet-fed larvae (controls) relative to their food source was markedly less (1.7 ± 0.1‰) than was observed in wood-fed larvae, yet still exceeded the threshold of 1‰. A predictive model based on δ(13)CEAA signatures of five EAAs from representative bacterial, fungal, and plant samples identified symbiotic bacteria and fungi as the likely supplementary sources of EAA in wood-fed larvae. Using the same model, but with an artificial diet as the dietary source, we identified minor supplementary bacterial sources of EAA in artificial diet-fed larvae. This study highlights how microbes associated with A. glabripennis can serve as a source of EAAs when fed on nutrient-limited diets, potentially circumventing the dietary limitations of feeding on woody substrates. PMID:26396228

  8. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

    PubMed

    Ichi, Ikuyo; Kono, Nozomu; Arita, Yuka; Haga, Shizuka; Arisawa, Kotoko; Yamano, Misato; Nagase, Mana; Fujiwara, Yoko; Arai, Hiroyuki

    2014-01-01

    In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9. PMID:24184513

  9. Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats.

    PubMed

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Mori, Masato; Takahashi, Michio

    2009-03-01

    It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine. PMID:18584286

  10. Essential Amino Acids in the Gluten-Free Diet and Serum in Relation to Depression in Patients with Celiac Disease

    PubMed Central

    van Hees, Nathalie J. M.; Giltay, Erik J.; Tielemans, Susanne M. A. J.; Geleijnse, Johanna M.; Puvill, Thomas; Janssen, Nadine; van der Does, Willem

    2015-01-01

    Introduction Celiac disease (CD) is associated with an increased risk of major depressive disorder, possibly due to deficiencies in micronutrients in the gluten-free diet. We aimed to investigate whether essential amino acids (i.e., the precursors of serotonin, dopamine and other neurotransmitters) are depleted in the diet and serum of CD patients with major depressive disorder. Methods In a cross-sectional study we assessed dietary intake of amino acids and serum levels of amino acids, in 77 CD patients on a gluten-free diet and in 33 healthy controls. Major depressive disorder was assessed with structured interviews (using the Mini International Neuropsychiatric Interview Plus). Dietary intake was assessed using a 203-item food frequency questionnaire. Results Participants had a mean age of 55 years and 74% were women. The intake of vegetable protein was significantly lower in CD patients than in healthy controls (mean difference of 7.8 g/d; 95% CI: 4.7–10.8), as were serum concentrations of tyrosine, phenylalanine and tryptophan (all p < 0.005). However, within the CD patient group, the presence of major depressive disorder (n = 42) was not associated with intake or serum levels of essential amino acids. Conclusions Patients with CD on a long-term gluten-free diet, with good adherence, consume significantly less vegetable protein than controls, and their serum levels of several essential amino acids were also lower. Despite its potential adverse effect, intake and serum levels of essential amino acids were not related to major depression. PMID:25884227

  11. Growth responses of breast and leg muscles to essential amino acids in broiler chicks.

    PubMed

    Mehri, M; Bagherzadeh-Kasmani, F; Rokouei, M

    2016-03-01

    The first three essential amino acids (EAA) for broilers including methionine (Met), lysine (Lys) and threonine (Thr) may greatly influence the growth of chick muscles at early stages of life. In order to survey the potential effects of those EAA on growth muscles, a rotatable three-variable central composite design (CCD) was conducted to track the interrelationships of dietary digestible Met (dMet), Lys (dLys) and Thr (dThr) for optimization of processing yields in broiler chicks using response surface methodology. A total of 60 floor pens of six birds each were assigned to 15 dietary treatments based on CCD containing five levels of dMet (0.416% to 0.584% of diet), dLys (0.881% to 1.319% of diet) and dThr (0.532% to 0.868% of diet) from 3 to 16 days of age. Experimental treatments significantly affected breast mass (BM) and leg mass (LM) of the birds (P<0.05) in which the main effect of dLys on BM was threefold higher than the main effect of dThr, and interaction effect between dMet and dLys was observed on BM (P<0.05). However, in the case of LM, the main effect of dThr was higher than the main effects of dMet and dLys and highest interaction effect exist between dThr and dMet (P<0.05). The second-order models for BM and LM were fitted by least squares regression. Canonical analysis revealed that the stationary points for carcass components were saddle points, thus ridge analysis was performed for getting optimal values of each EAA. Ridge analyses of BM and LM models showed that the maximum BM point may be obtained with 0.58%, 1.05% and 0.76% of dMet, dLys and dThr, respectively, in diet, and maximum LM point may be achieved with 0.58%, 1.09% and 0.70% of dMet, dLys and dThr, respectively, in diet. The resultant ideal ratios of dMet and dThr to dLys were 55% and 72% for BM; 53% and 64% for LM. Moreover, sensitivity analysis showed that the most important amino acids in BM and LM models were Lys and Thr, respectively. In conclusion, providing these three amino

  12. Polyunsaturated fatty acids inhibit Kv1.4 by interacting with positively charged extracellular pore residues.

    PubMed

    Farag, N E; Jeong, D; Claydon, T; Warwicker, J; Boyett, M R

    2016-08-01

    Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K(+) channel inactivation by an unknown site and mechanism. The effects of ω-6 and ω-3 PUFAs were investigated on the heterologously expressed Kv1.4 channel. PUFAs inhibited wild-type Kv1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant Kv1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (Kd, 15-43 μM). C-type inactivation was affected by extracellular H(+) and K(+) as well as PUFAs and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K(+) Replacement of two positively charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H(+) and K(+)) on C-type inactivation but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pKa of H508 and this reduces the K(+) occupancy of the selectivity filter, stabilizing the C-type inactivated state. PMID:27281482

  13. Degradation of carbohydrates during dilute sulfuric acid pretreatment can interfere with lignin measurements in solid residues.

    PubMed

    Katahira, Rui; Sluiter, Justin B; Schell, Daniel J; Davis, Mark F

    2013-04-01

    The lignin content measured after dilute sulfuric acid pretreatment of corn stover indicates more lignin than could be accounted for on the basis of the untreated corn stover lignin content. This phenomenon was investigated using a combination of (13)C cross-polarization/magic-angle spinning (CP/MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy and lignin removal using acid chlorite bleaching. Only minimal contamination with carbohydrates and proteins was observed in the pretreated corn stover. Incorporating degradation products from sugars was also investigated using (13)C-labeled sugars. The results indicate that sugar degradation products are present in the pretreatment residue and may be intimately associated with the lignin. Studies comparing whole corn stover (CS) to extractives-free corn stover [CS(Ext)] clearly demonstrated that extractives are a key contributor to the high-lignin mass balance closure (MBC). Sugars and other low molecular weight compounds present in plant extractives polymerize and form solids during pretreatment, resulting in apparent Klason lignin measurements that are biased high. PMID:23428141

  14. Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

    Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, C(α) and C(β) atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of 'hard' degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available

  15. SeqX: a tool to detect, analyze and visualize residue co-locations in protein and nucleic acid structures

    PubMed Central

    Biro, Jan C; Fördös, Gergely

    2005-01-01

    Background The interacting residues of protein and nucleic acid sequences are close to each other – they are co-located. Structure databases (like Protein Data Bank, PDB and Nucleic Acid Data Bank, NDB) contain all information about these co-locations; however it is not an easy task to penetrate this complex information. We developed a JAVA tool, called SeqX for this purpose. Results SeqX tool is useful to detect, analyze and visualize residue co-locations in protein and nucleic acid structures. The user a. selects a structure from PDB; b. chooses an atom that is commonly present in every residues of the nucleic acid and/or protein structure(s) c. defines a distance from these atoms (3–15 Å). The SeqX tool detects every residue that is located within the defined distances from the defined "backbone" atom(s); provides a DotPlot-like visualization (Residues Contact Map), and calculates the frequency of every possible residue pairs (Residue Contact Table) in the observed structure. It is possible to exclude +/- 1 to 10 neighbor residues in the same polymeric chain from detection, which greatly improves the specificity of detections (up to 60% when tested on dsDNA). Results obtained on protein structures showed highly significant correlations with results obtained from literature (p < 0.0001, n = 210, four different subsets). The co-location frequency of physico-chemically compatible amino acids is significantly higher than is calculated and expected in random protein sequences (p < 0.0001, n = 80). Conclusion The tool is simple and easy to use and provides a quick and reliable visualization and analyses of residue co-locations in protein and nucleic acid structures. Availability and requirements SeqX, Java J2SE Runtime Environment 5.0 (available from [see Additional file 1] ) and at least a 1 GHz processor and with a minimum 256 Mb RAM. Source codes are available from the authors. PMID:16011796

  16. Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis.

    PubMed Central

    Tamahara, Satoshi; Inaba, Mutsumi; Sato, Kota; Matsuki, Naoaki; Hikasa, Yoshiaki; Ono, Ken-Ichiro

    2002-01-01

    A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na(+)-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg(2+) and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters. PMID:12088508

  17. Intra-molecular cross-linking of acidic residues for protein structure studies.

    PubMed

    Novak, Petr; Kruppa, Gary H

    2008-01-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry

  18. Nonenzymatic browning reaction of essential amino acids: effect of pH on caramelization and Maillard reaction kinetics.

    PubMed

    Ajandouz, E H; Puigserver, A

    1999-05-01

    The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods. PMID:10552453

  19. Comparative evaluation of essential fatty acid composition of mothers' milk of some urban and suburban regions of West Bengal, India.

    PubMed

    Roy, Susmita; Dhar, Pubali; Ghosh, Santinath

    2012-12-01

    This study investigated the fatty acid composition of lipid present in breast milk of mothers residing in urban and suburban regions of West Bengal with special emphasis on n-6 and n-3 polyunsaturated fatty acids, which played a crucial role in the growth and development of neonates. Milk samples collected from 135 mothers of middle income group (average monthly income around 'Rs 10,000/-') were analysed by gas liquid chromatography after extraction and transmethylation to determine fatty acid composition. Information about the dietary intake of individual mothers was obtained through food frequency questionnaire. The fractions of n-6 and n-3 polyunsaturated fatty acids available in milk of urban mothers were 13.59 ± 0.94 and 3.65 ± 0.49, respectively, and in suburban mothers 12.74 ± 0.89 and 4.36 ± 0.39, respectively. The green leafy vegetables, fishes and vegetable oils were the major sources of essential fatty acids in the diet of the experimental groups of Bengali mothers. This study revealed a relationship between the alimentary habits of mothers and the concentration of essential fatty acids in breast milk of Bengali mothers. PMID:22575018

  20. Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin.

    PubMed

    Chan, Linda J; Rosengren, K Johan; Layfield, Sharon L; Bathgate, Ross A D; Separovic, Frances; Samuel, Chrishan S; Hossain, Mohammed A; Wade, John D

    2012-11-30

    Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin. PMID:23024363

  1. Predicting HLA Class I Non-Permissive Amino Acid Residues Substitutions

    PubMed Central

    Binkowski, T. Andrew; Marino, Susana R.; Joachimiak, Andrzej

    2012-01-01

    Prediction of peptide binding to human leukocyte antigen (HLA) molecules is essential to a wide range of clinical entities from vaccine design to stem cell transplant compatibility. Here we present a new structure-based methodology that applies robust computational tools to model peptide-HLA (p-HLA) binding interactions. The method leverages the structural conservation observed in p-HLA complexes to significantly reduce the search space and calculate the system’s binding free energy. This approach is benchmarked against existing p-HLA complexes and the prediction performance is measured against a library of experimentally validated peptides. The effect on binding activity across a large set of high-affinity peptides is used to investigate amino acid mismatches reported as high-risk factors in hematopoietic stem cell transplantation. PMID:22905104

  2. Potential of essential fatty acid deficiency with extremely low fat diet in lipoprotein lipase deficiency during pregnancy: A case report

    PubMed Central

    Tsai, Elaine C; Brown, Judy A; Veldee, Megan Y; Anderson, Gregory J; Chait, Alan; Brunzell, John D

    2004-01-01

    Background Pregnancy in patients with lipoprotein lipase deficiency is associated with high risk of maternal pancreatitis and fetal death. A very low fat diet (< 10% of calories) is the primary treatment modality for the prevention of acute pancreatitis, a rare but potentially serious complication of severe hypertriglyceridemia. Since pregnancy can exacerbate hypertriglyceridemia in the genetic absence of lipoprotein lipase, a further reduction of dietary fat intake to < 1–2% of total caloric intake may be required during the pregnancy, along with the administration of a fibrate. It is uncertain if essential fatty acid deficiency will develop in the mother and fetus with this extremely low fat diet, or whether fibrates will cross the placenta and concentrate in the fetus. Case presentation A 23 year-old gravida 1 woman with primary lipoprotein lipase deficiency was seen at 7 weeks of gestation in the Lipid Clinic for management of severe hypertriglyceridemia that had worsened with pregnancy. While on her habitual fat intake of 10% of total calories, her pregnancy resulted in an exacerbation of the hypertriglyceridemia, which prompted further restriction of fat intake to < 2% of total calories, as well as administration of gemfibrozil at a lower than average dose. The level of gemfibrozil, as the active metabolite, in the venous and arterial fetal cord blood was within the expected therapeutic range for adults. The clinical signs and a biomarker of essential fatty acid deficiency, namely the ratio of 20:3 [n-9] to 20:4 [n-6] fatty acids, were closely monitored throughout her pregnancy. Despite her extremely low fat diet, the levels of essential fatty acids measured in the mother and in the fetal blood immediately postpartum were normal. Normal essential fatty acid levels may have been achieved by the topical application of sunflower oil. Conclusions An extremely low fat diet in combination with topical sunflower oil and gemfibrozil administration was safely

  3. Chemical composition and antibacterial properties of essential oil and fatty acids of different parts of Ligularia persica Boiss

    PubMed Central

    Mohadjerani, Maryam; Hosseinzadeh, Rahman; Hosseini, Maryam

    2016-01-01

    Objective: The objective of this research was to investigate the chemical composition and antibacterial activities of the fatty acids and essential oil from various parts of Ligularia persica Boiss (L. persica) growing wild in north of Iran. Materials and Methods: Essential oils were extracted by using Clevenger-type apparatus. Antibacterial activity was tested on two Gram-positive and two Gram-negative bacteria by using micro dilution method. Results: GC and GC∕MS analysis of the oils resulted in detection of 94%, 96%, 93%, 99% of the total essential oil of flowers, stems, roots and leaves, respectively. The main components of flowers oil were cis-ocimene (15.4%), β-myrcene (4.4%), β-ocimene (3.9%), and γ-terpinene (5.0%). The major constituents of stems oil were β-phellandrene (5.4%), β-cymene (7.0%), valencene (3.9%). The main compounds of root oil were fukinanolid (17.0%), α-phellandrene (11.5%) and Β-selinene (5.0%) and in the case of leaves oil were cis-ocimene (4.8%), β-ocimene (4.9%), and linolenic acid methyl ester (4.7%). An analysis by GC-FID and GC-MS on the fatty-acid composition of the different parts of L. persica showed that major components were linoleic acid (11.3-31.6%), linolenic acid (4.7-21.8%) and palmitic acid (7.2-23.2%). Saturated fatty acids were found in lower amounts than unsaturated ones. The least minimum inhibition concentration (MIC) of the L. persica was 7.16 μg/ml against Pseudomonas aeruginosa. Conclusion: Our study indicated that the essential oil from L. persica stems and flowers showed high inhibitory effect on the Gram negative bacteria. The results also showed that fatty acids from the stems and leaves contained a high amount of poly-unsaturated fatty acids (PUFAs). PMID:27462560

  4. Complex formation of cadmium with sugar residues, nucleobases, phosphates, nucleotides, and nucleic acids.

    PubMed

    Sigel, Roland K O; Skilandat, Miriam; Sigel, Astrid; Operschall, Bert P; Sigel, Helmut

    2013-01-01

    Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand

  5. Conserved Glycine Residues in the Cytoplasmic Domain of the Aspartate Receptor Play Essential Roles in Kinase Coupling and On–Off Switching†

    PubMed Central

    Coleman, Matthew D.; Bass, Randal B.; Mehan, Ryan S.; Falke, Joseph J.

    2010-01-01

    The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on–off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on–off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on–off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on–off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends

  6. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues.

    PubMed

    Cook, Naomi L; Moeke, Cassidy H; Fantoni, Luca I; Pattison, David I; Davies, Michael J

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation. PMID:26616646

  7. Long-term effects of perinatal essential fatty acid deficiency on anxiety-related behavior in mice.

    PubMed

    Palsdottir, Vilborg; Månsson, Jan-Eric; Blomqvist, Maria; Egecioglu, Emil; Olsson, Bob

    2012-04-01

    Dietary essential fatty acids have been shown to regulate behavioral and cognitive functions in rodents. However, the long-term effect on behavior, besides memory and learning, of essential fatty acid deficiency (EFAD), i.e., lack of n-3 and n-6 fatty acids, during the perinatal period has not been investigated. Therefore, pregnant C57Bl/6 mice were given either an EFAD or an isoenergetic control diet from gestational day 16 and throughout lactation. The female offspring were given standard chow from 3 weeks of age, and at 12 to 14 weeks of age, open-field, object recognition, light-dark transition, elevated plus maze, and social interaction tests were performed. The brain glycerophospholipid fatty acid composition was investigated in 3-week-old and adult offspring by gas chromatography. The differences observed in behavior were indicative of lower anxiety in the EFAD mice compared to controls illustrated by more time spent in the open arms of the elevated plus maze (+ 41%, p < .05) and in the light compartment in the light-dark transition test (+ 63%, p < .05). The proportion of total n-3 fatty acids, especially 22:6n-3 in the brain, was lower with a compensatory increase in the proportion of total n-6 fatty acids, foremost 22:5n-6, in the EFAD mice compared to controls at 3 weeks of age. In the adult brains the fatty acid composition was normalized. In conclusion, our data show that EFAD during the perinatal period results in short-term alterations of fatty acid composition in brain and decreased anxiety in adult life. PMID:22352789

  8. AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

    PubMed Central

    Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

    2010-01-01

    AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

  9. Effect of second coagulant addition on coagulation efficiency, floc properties and residual Al for humic acid treatment by Al13 polymer and polyaluminum chloride (PACl).

    PubMed

    Xu, Weiying; Gao, Baoyu; Wang, Yan; Yue, Qinyan; Ren, Haijing

    2012-05-15

    Influence of second dose on coagulation efficiency, floc re-growth, fractal structure and residual Al of the effluent in humic acid (HA) coagulation with Al(13) polymer ([Al(13)O(4)(OH)(24)(H(2)O)(12)](7+)) and PACl were comparatively investigated in this study. Effects of breakage shear on the floc properties generated in the coagulation with and without additional dose were also investigated. The results indicated that additional dose during breakage could essentially improve the HA removal efficiency and floc re-growth in both Al(13) and PACl coagulations. Second doses of Al(13) at 0.5 and 1.0mg/L resulted in better turbidity and UV(254) removal as well as floc re-growth rather than higher additional dose of 1.5 and 2.0mg/L; while in PACl coagulation, more efficient HA removal and better floc re-growth were obtained at higher additional doses (1.0, 1.5 and 2.0mg/L). Small additional Al(13) could apparently increase the D(f) of re-formed flocs while the additional PACl displayed inconspicuous effect on floc D(f). The additional coagulant dose could alleviate the further decrease of re-grown floc size with increased breakage shear for both coagulants. The residual Al analysis implied that two-stage addition contributed to lower residual Al in effluent than one-time addition mode with the same total coagulant concentration. PMID:22410719

  10. Distributions of amino acids suggest that certain residue types more effectively determine protein secondary structure.

    PubMed

    Saraswathi, S; Fernández-Martínez, J L; Koliński, A; Jernigan, R L; Kloczkowski, A

    2013-10-01

    Exponential growth in the number of available protein sequences is unmatched by the slower growth in the number of structures. As a result, the development of efficient and fast protein secondary structure prediction methods is essential for the broad comprehension of protein structures. Computational methods that can efficiently determine secondary structure can in turn facilitate protein tertiary structure prediction, since most methods rely initially on secondary structure predictions. Recently, we have developed a fast learning optimized prediction methodology (FLOPRED) for predicting protein secondary structure (Saraswathi et al. in JMM 18:4275, 2012). Data are generated by using knowledge-based potentials combined with structure information from the CATH database. A neural network-based extreme learning machine (ELM) and advanced particle swarm optimization (PSO) are used with this data to obtain better and faster convergence to more accurate secondary structure predicted results. A five-fold cross-validated testing accuracy of 83.8 % and a segment overlap (SOV) score of 78.3 % are obtained in this study. Secondary structure predictions and their accuracy are usually presented for three secondary structure elements: α-helix, β-strand and coil but rarely have the results been analyzed with respect to their constituent amino acids. In this paper, we use the results obtained with FLOPRED to provide detailed behaviors for different amino acid types in the secondary structure prediction. We investigate the influence of the composition, physico-chemical properties and position specific occurrence preferences of amino acids within secondary structure elements. In addition, we identify the correlation between these properties and prediction accuracy. The present detailed results suggest several important ways that secondary structure predictions can be improved in the future that might lead to improved protein design and engineering. PMID:23907551

  11. Catalytic mechanism of S-type phycobiliprotein lyase: chaperone-like action and functional amino acid residues.

    PubMed

    Kupka, Michaela; Zhang, Juan; Fu, Wei-Lei; Tu, Jun-Ming; Böhm, Stephan; Su, Ping; Chen, Yu; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong

    2009-12-25

    The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300-14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-beta84-bound PCB in biliproteins by PEB. PMID:19864423

  12. Reclamation of acidic mine residues by creation of technosoils with the addition of biochar and marble waste

    NASA Astrophysics Data System (ADS)

    Moreno-Barriga, Fabián; Díaz, Vicente; Acosta, José; Faz, Ángel; Zornoza, Raul

    2016-04-01

    This study reports the short-term effect of biochar and marble waste addition for the reclamation of acidic mine residues. A lab incubation was carried out for 90 days. Biochars derived from pig manure (PM), crop residues (CR) and municipal solid waste (MSW) were added to the soil at a rate of 20 g kg-1. The marble waste (MW) was added at a rate of 200 g kg-1. Bochars and MW were applied independently and combined. A control soil was used without application of amendments. The evolution of different physical, chemical and biochemical properties and availability of heavy metals was periodically monitored. Results showed that original pH (2.8) was increased with all amendments, those samples containing MW being the ones with the highest pH (~8.0). The electrical conductivity (EC) decreased from 6.6 to 3.0-4.5 mS cm-1 in all the treatments receiving MW. Soil organic C (SOC) increased in all samples receiving biochar up to 18-20 g kg-1, with no shifts during the 90 d incubation, indicating the high stability of the C supplied. Recalcitrant organic C accounted for ~90-98% of the SOC. No significant effect of amendment addition was observed for carbohydrates, soluble C, microbial biomass C and β-glucosidase activity. However, arylesterase activity increased with amendments, highly related to pH. The availability of heavy metals decreased up to 90-95% owing to the addition of amendments, mainly in samples containing MW. The MW provided conditions to increase pH and decrease EC and metals mobility. Biochar was an effective strategy to increase SOC, recalcitrant C and AS, essential to create soil structure. However, a labile source of organic matter should be added together with the proposed amendments to promote the activation of microbial communities. Acknowledgement : This work has been funded by Fundación Séneca (Agency of Science and Technology of the Region of Murcia, Spain) by the project 18920/JLI/13

  13. D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

    PubMed

    Torres, Allan M; Tsampazi, Chryssanthi; Geraghty, Dominic P; Bansal, Paramjit S; Alewood, Paul F; Kuchel, Philip W

    2005-10-15

    The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution. PMID:16033333

  14. Preparation of a novel carbon-based solid acid from cassava stillage residue and its use for the esterification of free fatty acids in waste cooking oil.

    PubMed

    Wang, Lingtao; Dong, Xiuqin; Jiang, Haoxi; Li, Guiming; Zhang, Minhua

    2014-04-01

    A novel carbon-based solid acid catalyst was prepared by the sulfonation of incompletely carbonized cassava stillage residue (CSR) with concentrated sulfuric acid, and employed to catalyze the esterification of methanol and free fatty acids (FFAs) in waste cooking oil (WCO). The effects of the carbonization and the sulfonation temperatures on the pore structure, acid density and catalytic activity of the CSR-derived catalysts were systematically investigated. Low temperature carbonization and high temperature sulfonation can cause the collapse of the carbon framework, while high temperature carbonization is not conducive to the attachment of SO3H groups on the surface. The catalyst showed high catalytic activity for esterification, and the acid value for WCO is reduced to below 2mg KOH/g after reaction. The activity of catalyst can be well maintained after five cycles. CSR can be considered a promising raw material for the production of a new eco-friendly solid acid catalyst. PMID:24661813

  15. Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol.

    PubMed

    Tan, Inn Shi; Lee, Keat Teong

    2015-06-25

    The aim of this study is to investigate the technical feasibility of converting macroalgae cellulosic residue (MCR) into bioethanol. An attempt was made to present a novel, environmental friendly and economical pretreatment process that enhances enzymatic conversion of MCR to sugars using Dowex (TM) Dr-G8 as catalyst. The optimum yield of glucose reached 99.8% under the optimal condition for solid acid pretreatment (10%, w/v biomass loading, 4%, w/v catalyst loading, 30min, 120°C) followed by enzymatic hydrolysis (45FPU/g of cellulase, 52CBU/g of β-glucosidase, 50°C, pH 4.8, 30h). The yield of sugar obtained was found more superior than conventional pretreatment process using H2SO4 and NaOH. Biomass loading for the subsequent simultaneous saccharification and fermentation (SSF) of the pretreated MCR was then optimized, giving an optimum bioethanol yield of 81.5%. The catalyst was separated and reused for six times, with only a slight drop in glucose yield. PMID:25839825

  16. Spatial Patterns and Temperature Predictions of Tuna Fatty Acids: Tracing Essential Nutrients and Changes in Primary Producers.

    PubMed

    Pethybridge, Heidi R; Parrish, Christopher C; Morrongiello, John; Young, Jock W; Farley, Jessica H; Gunasekera, Rasanthi M; Nichols, Peter D

    2015-01-01

    Fatty acids are among the least understood nutrients in marine environments, despite their profile as key energy components of food webs and that they are essential to all life forms. Presented here is a novel approach to predict the spatial-temporal distributions of fatty acids in marine resources using generalized additive mixed models. Fatty acid tracers (FAT) of key primary producers, nutritional condition indices and concentrations of two essential long-chain (≥C20) omega-3 fatty acids (EFA) measured in muscle of albacore tuna, Thunnus alalunga, sampled in the south-west Pacific Ocean were response variables. Predictive variables were: location, time, sea surface temperature (SST) and chlorophyll-a (Chla), and phytoplankton biomass at time of catch and curved fork length. The best model fit for all fatty acid parameters included fish length and SST. The first oceanographic contour maps of EFA and FAT (FATscapes) were produced and demonstrated clear geographical gradients in the study region. Predicted changes in all fatty acid parameters reflected shifts in the size-structure of dominant primary producers. Model projections show that the supply and availability of EFA are likely to be negatively affected by increases in SST especially in temperate waters where a 12% reduction in both total fatty acid content and EFA proportions are predicted. Such changes will have large implications for the availability of energy and associated health benefits to high-order consumers. Results convey new concerns on impacts of projected climate change on fish-derived EFA in marine systems. PMID:26135308

  17. Spatial Patterns and Temperature Predictions of Tuna Fatty Acids: Tracing Essential Nutrients and Changes in Primary Producers

    PubMed Central

    Pethybridge, Heidi R.; Parrish, Christopher C.; Morrongiello, John; Young, Jock W.; Farley, Jessica H.; Gunasekera, Rasanthi M.; Nichols, Peter D.

    2015-01-01

    Fatty acids are among the least understood nutrients in marine environments, despite their profile as key energy components of food webs and that they are essential to all life forms. Presented here is a novel approach to predict the spatial-temporal distributions of fatty acids in marine resources using generalized additive mixed models. Fatty acid tracers (FAT) of key primary producers, nutritional condition indices and concentrations of two essential long-chain (≥C20) omega-3 fatty acids (EFA) measured in muscle of albacore tuna, Thunnus alalunga, sampled in the south-west Pacific Ocean were response variables. Predictive variables were: location, time, sea surface temperature (SST) and chlorophyll-a (Chla), and phytoplankton biomass at time of catch and curved fork length. The best model fit for all fatty acid parameters included fish length and SST. The first oceanographic contour maps of EFA and FAT (FATscapes) were produced and demonstrated clear geographical gradients in the study region. Predicted changes in all fatty acid parameters reflected shifts in the size-structure of dominant primary producers. Model projections show that the supply and availability of EFA are likely to be negatively affected by increases in SST especially in temperate waters where a 12% reduction in both total fatty acid content and EFA proportions are predicted. Such changes will have large implications for the availability of energy and associated health benefits to high-order consumers. Results convey new concerns on impacts of projected climate change on fish-derived EFA in marine systems. PMID:26135308

  18. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    PubMed Central

    Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Ching-Ting; Ho, Tin-Yun; Hsiang, Chien-Yun

    2009-01-01

    Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. PMID:19272175

  19. Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions

    PubMed Central

    Smerilli, Marina; Neureiter, Markus; Wurz, Stefan; Haas, Cornelia; Frühauf, Sabine; Fuchs, Werner

    2015-01-01

    BACKGROUND Lactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs. RESULTS A process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3–l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L−1 optically pure (98%) L-lactic acid in 20 h from 50 g L−1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch. CONCLUSIONS Potato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25937690

  20. An in vitro model for essential fatty acid deficiency: HepG2 cells permanently maintained in lipid-free medium.

    PubMed

    Furth, E E; Sprecher, H; Fisher, E A; Fleishman, H D; Laposata, M

    1992-11-01

    A stable essential fatty acid-deficient cell type, known as HepG2-EFD, was derived from the lipoprotein-producing human hepatoma cell line HepG2. These cells are particularly useful for quantitative studies involving essential fatty acids (n-6 and n-3 fatty acids) in secreted lipoproteins. Radiolabeled essential fatty acids can be delivered to these cells without altering the specific activity of the fatty acids, since the deficient cells contain no endogenous essential fatty acids. Using these cells, radioactivity data (dpm) from metabolic studies can be converted directly to mass, and masses as low as a few pmoles can be accurately measured. HepG2-EFD cell cultures were established by growing HepG2 cells in medium containing delipidated serum. After 10 days of growth in delipidated medium, HepG2 cells were completely depleted of all essential fatty acids. Compensatory increases in nonessential fatty acids (n-9 and n-7 fatty acids) including 20:3n-9 (the Mead acid), which is the hallmark fatty acid of essential fatty acid deficiency, were also observed in HepG2-EFD cells. Despite the lack of exogenous fatty acids in the medium and the lack of essential fatty acids in the cells, export of very low density lipoprotein (VLDL)-associated apolipoprotein B by HepG2-EFD was the same as observed for parent HepG2 cells. However, the activity of beta-oxidation of fatty acids in HepG2-EFD cells was much lower than in the parent cell line.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1464755

  1. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    PubMed

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  2. Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

    PubMed Central

    Wang, Kun; Tian, Jian; Turunen, Ossi; Huang, Huoqing; Shi, Pengjun; Hua, Huifang; Wang, Caihong; Wang, Shuanghe

    2014-01-01

    Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability. PMID:24463976

  3. Simultaneous Determination of Essential Oil Components and Fatty Acids in Fennel using Gas Chromatography with a Polar Capillary Column.

    PubMed

    Najdoska-Bogdanov, Menče; Bogdanov, Jane B; Stefova, Marina

    2015-09-01

    Cultivated and wild growing samples of fennel (Foeniculum vulgare Mill., Apiaceae) from R. Macedonia were studied for their volatiles and fatty acid composition. The main essential oil components isolated via hydrodistillation were: trans-anethole (>80%), estragole (< 6%), limonene (< 6%), anisaldehyde (< 1%) and 0.5 % fenchone. An alternative method for characterization of both the non-polar volatile and non volatile fractions was developed using n-hexane and dichloromethane (3:1, v/v) in a Soxhlet extraction followed by transesterification. The obtained extracts were then characterized and the dominant fatty acid was 18:1 (petroselinic and oleic acid) 75.0-82.8%, followed by 18:2 (linoleic acid) 10.8-16.2% and other fatty acids: palmitic (4.3-6.9%), stearic (1.2-1.7%) and myristic (0-2.9%). The results for the volatile fraction after Soxhlet extraction and transesterification did not significantly differ from results obtained after hydrodistillation, especially for the main components (trans-anethole, estragole, fenchone and limonene), implying that the developed method can be used for simultaneous determination of volatiles and fatty acids. PMID:26594773

  4. Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter Pylori

    SciTech Connect

    Schoenhofen,I.; Lunin, V.; Julien, J.; Li, Y.; Ajamian, E.; Matte, A.; Cygler, M.; Brisson, J.; Aubry, A.; et al.

    2006-01-01

    Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. As motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-a-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-{beta}-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc product, the latter being converted to the external aldimine form in the enzyme's active site. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the {beta}-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and in conjunction with the recent functional characterization of this enzyme, will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  5. The Hip Functional Retrieval after Elective Surgery May Be Enhanced by Supplemented Essential Amino Acids

    PubMed Central

    Baldissarro, Eleonora; Aquilani, Roberto; Boschi, Federica; Baiardi, Paola; Iadarola, Paolo; Fumagalli, Marco; Pasini, Evasio; Verri, Manuela; Dossena, Maurizia; Gambino, Arianna; Cammisuli, Sharon; Viglio, Simona

    2016-01-01

    It is not known whether postsurgery systemic inflammation and plasma amino acid abnormalities are still present during rehabilitation of individuals after elective hip arthroplasty (EHA). Sixty subjects (36 females; age 66.58 ± 8.37 years) were randomized to receive 14-day oral EAAs (8 g/day) or a placebo (maltodextrin). At admission to and discharge from the rehabilitation center, serum C-reactive protein (CRP) and venous plasma amino acid concentrations were determined. Post-EHA hip function was evaluated by Harris hip score (HHS) test. Ten matched healthy subjects served as controls. At baseline, all patients had high CRP levels, considerable reduction in several amino acids, and severely reduced hip function (HHS 40.78 ± 2.70 scores). After treatment, inflammation decreased both in the EAA group and in the placebo group. Only EAA patients significantly improved their levels of glycine, alanine, tyrosine, and total amino acids. In addition, they enhanced the rate of hip function recovery (HHS) (from baseline 41.8 ± 1.15 to 76.37 ± 6.6 versus baseline 39.78 ± 4.89 to 70.0 ± 7.1 in placebo one; p = 0.006). The study documents the persistence of inflammation and plasma amino acid abnormalities in post-EHA rehabilitation phase. EAAs enhance hip function retrieval and improve plasma amino acid abnormalities. PMID:27110573

  6. A 13C{31P} REDOR NMR Investigation of the Role of Glutamic Acid Residues in Statherin-Hydroxyapatite Recognition

    PubMed Central

    Ndao, Moise; Ash, Jason T.; Breen, Nicholas F.; Goobes, Gil; Stayton, Patrick S.; Drobny, Gary P.

    2011-01-01

    The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids – D1, E4, and E5 – are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized 13C{31P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the δ-carboxyl 13C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and 31P spins of the phosphate groups at the HAP surface. 13C{31P} REDOR studies of glutamic-5-13C acid incorporated at positions E4 and E26 indicate a 13C–31P distance of more than 6.5 Å between the side chain carboxyl 13C spin of E4 and the closest 31P in the HAP surface. In contrast, the carboxyl 13C spin at E5 has a much shorter 13C–31P internuclear distance of 4.25±0.09 Å, indicating that the carboxyl group of this side chain interacts directly with the surface. 13C T1ρ and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces. PMID:19678690

  7. Functional analysis of ars gene cluster of Pannonibacter indicus strain HT23(T) (DSM 23407(T)) and identification of a proline residue essential for arsenate reductase activity.

    PubMed

    Bandyopadhyay, Saumya; Das, Subrata K

    2016-04-01

    Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC. PMID:26915994

  8. T Cell Determinants Incorporating [beta]-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

    SciTech Connect

    Webb, Andrew I.; Dunstone, Michelle A.; Williamson, Nicholas A.; Price, Jason D.; Kauwe, Andreade; Chen, Weisan; Oakley, Aaron; Perlmutter, Patrick; McCluskey, James; Aguilar, Marie-Isabel; Rossjohn, Jamie; Purcell, Anthony W.

    2010-07-20

    A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of {beta}-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding {beta}-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with {beta}-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using {beta}-amino acids. We conclude that the rational incorporation of {beta}-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring {alpha}-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

  9. BK channel activation by tungstate requires the β1 subunit extracellular loop residues essential to modulate voltage sensor function and channel gating.

    PubMed

    Fernández-Mariño, Ana I; Valverde, Miguel A; Fernández-Fernández, José M

    2014-07-01

    Tungstate, a compound with antidiabetic, antiobesity, and antihypertensive properties, activates the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel containing either β1 or β4 subunits. The BK activation by tungstate is Mg(2+)-dependent and promotes arterial vasodilation, but only in precontracted mouse arteries expressing β1. In this study, we further explored how the β1 subunit participates in tungstate activation of BK channels. Activation of heterologously expressed human BKαβ1 channels in inside-out patches is fully dependent on the Mg(2+) sensitivity of the BK α channel subunit even at high (10 μM) cytosolic Ca(2+) concentration. Alanine mutagenesis of β1 extracellular residues Y74 or S104, which destabilize the active voltage sensor, greatly decreased the tungstate-induced left-shift of the BKαβ1 G-V curves in either the absence or presence of physiologically relevant cytosolic Ca(2+) levels (10 μM). The weakened tungstate activation of the BKαβ1Y74A and BKαβ1S104A mutant channels was not related to decreased Mg(2+) sensitivity. These results, together with previously published reports, support the idea that the putative binding site for tungstate-mediated BK channel activation is located in the pore-forming α channel subunit, around the Mg(2+) binding site. The role of β1 in tungstate-induced channel activation seems to rely on its interaction with the BK α subunit to modulate channel activity. Loop residues that are essential for the regulation of voltage sensor activation and gating of the BK channel are also relevant for BK activation by tungstate. PMID:24158430

  10. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii.

    PubMed

    Martins-Duarte, Érica S; Carias, Maira; Vommaro, Rossiane; Surolia, Namita; de Souza, Wanderley

    2016-09-01

    The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of 'tethered' daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division. PMID:27457282

  11. Residual cadmium forms in acid-extracted anaerobically digested sewage sludge

    SciTech Connect

    Feltz, R.E.; Logan, T.J.

    1985-01-01

    The effects of phosphorus and lime additions after acid extraction on residual Cd solubility and chemical forms in an anaerobically digested sewage sludge were investigated. High Cd content anaerobically digested sewage sludge was aerated and then acidified to pH 2 to solubilize Cd. After 18 h of acidification, the sludge was dewatered and the supernatant and solids separated. Seventy or more percent of the Cd was removed from the solids. Similar amounts of Ni, Mn and Zn were also removed, but Cu removal was only 26% and that of Pb was < 5%. Before liming the sludge was amended with rock phosphate (RP) or monocalcium phosphate (MCP). The RP was insoluble while MCP dissolved, providing a high level of phosphate ligand for Cd precipitation or coprecipitation. Estimated sludge solution solubility products for major Fe, Al and Ca phosphates showed that several of these minerals could have precipitated with P addition, especially with MCP, and Cd may have coprecipitated with these solid phases. Cadmium phosphate may also have been formed in the MCP sludge. Chemical fractionation indicated that 50% of the Cd in the aerated unextracted sludge existed as inorganic precipitates with another 40% Na/sub 4/P/sub 2/O/sub 7/ extractable. Acidification solubilized 98% of the inorganic Cd and 86% of the organically bound Cd. Seventy-nine percent of the Cd remaining in the dewatered acidified sludge was in the KNO/sub 3/ extractable (exchangeable) fraction. Liming redistributed the Cd with 13 to 19% as inorganic precipitates, 70 to 85% organically bound and < 3% in the exchangeable fraction. Phosphate addition had no significant effect on Cd fractionation.

  12. Reuse of acid coagulant-recovered drinking waterworks sludge residual to remove phosphorus from wastewater

    NASA Astrophysics Data System (ADS)

    Yang, Lan; Wei, Jie; Zhang, Yumei; Wang, Jianli; Wang, Dongtian

    2014-06-01

    Acid coagulant-recovered drinking waterworks sludge residual (DWSR) is a waste product from drinking waterworks sludge (DWS) treatment with acid for coagulant recovery. In this study, we evaluated DWSR as a potential phosphorus (P) removing material in wastewater treatment by conducting a series of batch and semi-continuous tests. Batch tests were carried out to study the effects of pH, initial concentration, and sludge dose on P removal. Batch test results showed that the P removal efficiency of DWSR was highly dependent on pH. Calcinated DWSR (C-DWSR) performed better in P removal than DWSR due to its higher pH. At an optimum initial pH value of 5-6 and a sludge dose of 10 g/L, the P removal rates of DWSR and DWS decreased from 99% and 93% to 84% and 14%, respectively, and the specific P uptake of DWSR and DWS increased from 0.19 and 0.19 mg P/g to 33.60 and 5.72 mg P/g, respectively, when the initial concentration was increased from 2 to 400 mg/L. The effective minimum sludge doses of DWSR and DWS were 0.5 g/L and 10 g/L, respectively, when the P removal rates of 90% were obtained at an initial concentration of 10 mg/L. Results from semi-continuous test indicated that P removal rates over 99% were quickly achieved for both synthetic and actual wastewater (lake water and domestic sewage). These rates could be maintained over a certain time under a certain operational conditions including sludge dose, feed flow, and initial concentration. The physicochemical properties analysis results showed that the contents of aluminum (Al) and iron (Fe) in DWSR were reduced by 50% and 70%, respectively, compared with DWS. The insoluble Al and Fe hydroxide in DWS converted into soluble Al and Fe in DWSR. Metal leaching test results revealed that little soluble Al and Fe remained in effluent when DWSR was used for P removal. We deduced that chemical precipitation might be the major action for P removal by DWSR and that adsorption played only a marginal role.

  13. Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids

    PubMed Central

    Eoh, Hyungjin; Rhee, Kyu Y.

    2014-01-01

    Few mutations attenuate Mycobacterium tuberculosis (Mtb) more profoundly than deletion of its isocitrate lyases (ICLs). However, the basis for this attenuation remains incompletely defined. Mtb’s ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases required for growth on even and odd chain fatty acids. Here, we report that Mtb’s ICLs are essential for survival on both acetate and propionate because of its methylisocitrate lyase (MCL) activity. Lack of MCL activity converts Mtb’s methylcitrate cycle into a “dead end” pathway that sequesters tricarboxylic acid (TCA) cycle intermediates into methylcitrate cycle intermediates, depletes gluconeogenic precursors, and results in defects of membrane potential and intrabacterial pH. Activation of an alternative vitamin B12-dependent pathway of propionate metabolism led to selective corrections of TCA cycle activity, membrane potential, and intrabacterial pH that specifically restored survival, but not growth, of ICL-deficient Mtb metabolizing acetate or propionate. These results thus resolve the biochemical basis of essentiality for Mtb’s ICLs and survival on fatty acids. PMID:24639517

  14. Fatty Acid and Essential Oil Compositions of Trifolium angustifolium var. angustifolium with Antioxidant, Anticholinesterase and Antimicrobial Activities

    PubMed Central

    Ertaş, Abdulselam; Boğa, Mehmet; Haşimi, Nesrin; Yılmaz, Mustafa Abdullah

    2015-01-01

    This study represents the first report on the chemical composition and biological activity of Trifolium angustifolium var. angustifolium. The major components of the essential oil were identified as hexatriacontene (23.0%), arachidic acid (15.5%) and α-selinene (10.0%). The main constituents of the fatty acid obtained from the petroleum ether extract were identified as palmitic acid (29.8%), linoleic acid (18.6%) and oleic acid (10.5%). In particular, the water extract exhibited higher activity than α-tocopherol and BHT, which were used as standards in the ABTS cation radical scavenging assay and indicated higher inhibitory effect against acetylcholinesterase enzyme than the reference compound, galanthamine but exhibited weak activity in β-carotene bleaching, DPPH-free radical scavenging, and cupric-reducing antioxidant capacity assays. The petroleum ether extract exhibited higher activity than α-tocopherol which was used as standard in the β-carotene bleaching method at concentration 100 μg/mL. The acetone extract exhibited higher activity than α-tocopherol which was used as standard cupric reducing antioxidant capacity (CUPRAC) method at 100 μg/mL concentration. The acetone and methanol extracts were active on all microorganisms tested with a small zone diameter indicating weak activity. PMID:25561929

  15. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  16. Treatment of air pollution control residues with iron rich waste sulfuric acid: does it work for antimony (Sb)?

    PubMed

    Okkenhaug, Gudny; Breedveld, Gijs D; Kirkeng, Terje; Lægreid, Marit; Mæhlum, Trond; Mulder, Jan

    2013-03-15

    Antimony (Sb) in air pollution control (APC) residues from municipal solid waste incineration has gained increased focus due to strict Sb leaching limits set by the EU landfill directive. Here we study the chemical speciation and solubility of Sb at the APC treatment facility NOAH Langøya (Norway), where iron (Fe)-rich sulfuric acid (∼3.6M, 2.3% Fe(II)), a waste product from the industrial extraction of ilmenite, is used for neutralization. Antimony in water extracts of untreated APC residues occurred exclusively as pentavalent antimonate, even at low pH and Eh values. The Sb solubility increased substantially at pH<10, possibly due to the dissolution of ettringite (at alkaline pH) or calcium (Ca)-antimonate. Treated APC residues, stored anoxically in the laboratory, simulating the conditions at the NOAH Langøya landfill, gave rise to decreasing concentrations of Sb in porewater, occurring exclusively as Sb(V). Concentrations of Sb decreased from 87-918μgL(-1) (day 3) to 18-69μgL(-1) (day 600). We hypothesize that an initial sorption of Sb to Fe(II)-Fe(III) hydroxides (green rust) and eventually precipitation of Ca- and Fe-antimonates (tripuhyite; FeSbO4) occurred. We conclude that Fe-rich, sulfuric acid waste is efficient to immobilize Sb in APC residues from waste incineration. PMID:23465722

  17. THE CIRCADIAN CLOCK WITHIN THE CARDIOMYOCYTE IS ESSENTIAL FOR RESPONSIVENESS OF THE HEART TO FATTY ACIDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesi...

  18. An essential role of a ferritin-like protein in acid stress tolerance of Listeria monocytogenes.

    PubMed

    Milecka, Dorota; Samluk, Anna; Wasiak, Katarzyna; Krawczyk-Balska, Agata

    2015-03-01

    The expression of ten genes of Listeria monocytogenes previously identified as penicillin G-inducible was transcriptionally analyzed in the presence of 0.5 M KCl, pH 5.0 and 42 °C. This study revealed that all the genes are upregulated by osmotic stress, seven by acid stress and four by temperature stress conditions. The contribution of a gene encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcription regulator (axyR) to temperature, acid and osmotic stress tolerance was further examined by analysis of nonpolar deletion mutants. This revealed that a lack of PhoP or AxyR does not affect the ability to grow under the tested stress conditions. However, the Δ fri strain showed slightly delayed growth under osmotic and clearly impaired growth under acid stress conditions, indicating an important role of the ferritin-like protein in acid stress tolerance. PMID:25352185

  19. Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

    PubMed Central

    Elce, John S.

    1980-01-01

    Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[14C]acetamide. One of these possessed a carboxy group, which formed a [14C]glycollamide ester, and the other was cysteine, shown by isolation of S-[14C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[14C]acetamide. 4. Isolation of the γ-[14C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[14C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[14C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed. PMID:6104953

  20. Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

    PubMed Central

    Chen, Y; Sinha, K; Perumal, K; Reddy, R

    2000-01-01

    It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs. PMID:10999605

  1. Biosynthesis of D-alanyl-lipoteichoic acid by Lactobacillus casei: interchain transacylation of D-alanyl ester residues

    SciTech Connect

    Childs, W.C. 3d.; Taron, D.J.; Neuhaus, F.C.

    1985-06-01

    Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-(/sup 14/C)alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. The authors propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.

  2. Essential fatty acid supplementation during lactation is required to maximize the subsequent reproductive performance of the modern sow.

    PubMed

    Rosero, David S; Boyd, R Dean; McCulley, Mark; Odle, Jack; van Heugten, Eric

    2016-05-01

    This study was conducted to investigate the effects of supplemental essential fatty acids (EFA) on sow reproductive efficiency and to estimate the concentrations of EFA required by the lactating sow for maximum subsequent reproduction. Data were collected on 480 sows (PIC Camborough) balanced by parity, with 241 and 239 sows representing Parity 1, and 3-5 (P3+), respectively. Sows were assigned randomly, within parity, to a 3×3 factorial arrangement plus a control diet without added lipids. Factors included linoleic (2.1%, 2.7%, and 3.3%) and α-linolenic acid (0.15%, 0.30%, and 0.45%), obtained by adding 4% of different mixtures of canola, corn and flaxseed oils to diets. Diets were corn-soybean meal based with 12% wheat middlings. The benefits of supplemental EFA were more evident for the subsequent reproduction of mature P3+ sows. For these sows, supplemental α-linolenic acid improved the proportion of sows that farrowed relative to sows weaned (linear P=0.080; 82.8, 80.5, and 92.8% for sows fed 0.15%, 0.30%, and 0.45% α-linolenic acid, respectively). In addition, supplemental linoleic acid, fed to Parity 1 and P3+ sows, tended to increase subsequent litter size (linear P=0.074; 13.2, 13.8 and 14.0 total pigs born for 2.1%, 2.7% and 3.3% linoleic acid, respectively). These results demonstrate that a minimum dietary intake of both α-linolenic and linoleic acid is required for the modern lactating sow to achieve a maximum reproductive outcome through multiple mechanisms that include rapid return to estrus, increased maintenance of pregnancy and improved subsequent litter size. PMID:27037065

  3. Structure-function relationship of Vibrio harveyi NADPH-flavin oxidoreductase FRP: essential residues Lys167 and Arg15 for NADPH binding.

    PubMed

    Chung, Hae-Won; Tu, Shiao-Chun

    2012-06-19

    Vibrio harveyi NADPH-FMN oxidoreductase (FRP) catalyzes flavin reduction by NADPH. In comparing amino acid sequence and crystal structure with Escherichia coli NfsA, residues N134, R225, R133, K167, and R15 were targeted for investigation of their possible roles in the binding and utilization of the NADPH substrate. By mutation of each of these five residues to an alanine, steady-state rate analyses showed that the variants K167A and R15A had apparently greatly increased K(m,NADPH) and reduced k(cat)/K(m,NADPH), whereas little or much more modest changes were found for the other variants. The deuterium isotope effects (D)(V/K) for (4R)-[4-(2)H]-NADPH were markedly increased to 6.3 and 7.4 for K167A and R15A, respectively, indicating that the rate constants for NADPH and NADP(+) dissociation were greatly enhanced relative to the hydride transfer steps. Also, anaerobic stopped-flow analyses revealed that the equilibrium dissociation constant for NADPH binding (K(d)) to be 2.5-3.9 and 1.1 mM for K167A and R15A, respectively, much higher than the 0.4 μM K(d) for the native FRP, whereas the k(cat) of these two variants were similar to that of the wild-type enzyme. Moreover, the K167 to alanine mutation led to even a slight increase in k(cat)/K(m) for NADH. These results, taken together, provide a strong support to the conclusion that K167 and R15 each was critical in the binding of NADPH by FRP. Such a functional role may also exist for other FRP homologous proteins. PMID:22650604

  4. Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

    PubMed

    Ghatak, Chiranjib; Rodnin, Mykola V; Vargas-Uribe, Mauricio; McCluskey, Andrew J; Flores-Canales, Jose C; Kurnikova, Maria; Ladokhin, Alexey S

    2015-04-01

    The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. PMID:25875295

  5. A Review: Supplementation of Foods with Essential Fatty Acids-Can It Turn a Breeze without Further Ado?

    PubMed

    Ganesh, Vijayalakshmi; Hettiarachchy, Navam S

    2016-07-01

    This paper focuses on the critical aspects of supplementation of foods with essential fatty acids (EFAs), the need, health benefits of supplementation and the constraints of the process. Current trend of supplementation of foods with EFAs has been gaining momentum and more research pioneers due to the health benefits in par with the direct intake of EFA supplements. Technologies including encapsulation, nanotechnology, molecular complexing, genetic engineering and more emerging means, hold promise to food supplementation with EFAs. Food trials with adoption of various technologies, studies of bioavailability and health benefits are still underway and crucial before EFA supplementation in foods can hit the market on a global scale. PMID:26147443

  6. Bed rest impairs skeletal muscle amino acid transporter expression, mTORC1 signaling, and protein synthesis in response to essential amino acids in older adults

    PubMed Central

    Dickinson, Jared M.; Fry, Christopher S.; Walker, Dillon K.; Gundermann, David M.; Reidy, Paul T.; Timmerman, Kyle L.; Markofski, Melissa M.; Paddon-Jones, Douglas; Rasmussen, Blake B.; Volpi, Elena

    2012-01-01

    Skeletal muscle atrophy during bed rest is attributed, at least in part, to slower basal muscle protein synthesis (MPS). Essential amino acids (EAA) stimulate mammalian target of rapamycin (mTORC1) signaling, amino acid transporter expression, and MPS and are necessary for muscle mass maintenance, but there are no data on the effect of inactivity on this anabolic mechanism. We hypothesized that bed rest decreases muscle mass in older adults by blunting the EAA stimulation of MPS through reduced mTORC1 signaling and amino acid transporter expression in older adults. Six healthy older adults (67 ± 2 yr) participated in a 7-day bed rest study. We used stable isotope tracers, Western blotting, and real-time qPCR to determine the effect of bed rest on MPS, muscle mTORC1 signaling, and amino acid transporter expression and content in the postabsorptive state and after acute EAA ingestion. Bed rest decreased leg lean mass by ∼4% (P < 0.05) and increased postabsorptive mTOR protein (P < 0.05) levels while postabsorptive MPS was unchanged (P > 0.05). Before bed rest acute EAA ingestion increased MPS, mTOR (Ser2448), S6 kinase 1 (Thr389, Thr421/Ser424), and ribosomal protein S6 (Ser240/244) phosphorylation, activating transcription factor 4, L-type amino acid transporter 1 and sodium-coupled amino acid transporter 2 protein content (P < 0.05). However, bed rest blunted the EAA-induced increase in MPS, mTORC1 signaling, and amino acid transporter protein content. We conclude that bed rest in older adults significantly attenuated the EAA-induced increase in MPS with a mechanism involving reduced mTORC1 signaling and amino acid transporter protein content. Together, our data suggest that a blunted EAA stimulation of MPS may contribute to muscle loss with inactivity in older persons. PMID:22338078

  7. Dry heat popping of amaranth seed might damage some of its essential amino acids.

    PubMed

    Tovar, L R; Brito, E; Takahashi, T; Miyazawa, T; Soriano, J; Fujimoto, K

    1989-12-01

    Amaranth was a major crop among the Aztecs. In Mexico the seed is popped and eaten with brown sugar. The crude protein content of the seed is 14 +/- 2% but its contents of lysine and tryptophan are 6.2 and 1.6 g/16 g N respectively. We developed a popping method based on a fluid bed system (FBS) whereas the traditional method (TM) is just to pop the seeds manually in a hot plate. Assays carried out were evaluation of racemization of the amaranth protein due to heat treatment, amino acid composition of the raw and heat treated seeds and a biological experiment testing whether leucine was the most limiting amino acid of amaranth protein. Male rats were fed both popped amaranths and roasted amaranth. Parboiled amaranth and casein were controls. The results were: (a) Lys, Arg and Cys were damaged in the heat treated seeds; (b) Asp, Met, Glu, Ala and Phe were racemized in that decreasing order in the seeds popped and roasted by the TM; (c) the estimated net protein retention (NPR) and estimated net protein utilization (NPU) of popped amaranths by either method were not different, but were lower than for the parboiled amaranth. The parboiled amaranth was not different from casein; (d) Leu was not the most limiting amino acid in any of the amaranth seeds tested. After Lys, sulfur amino acids appear to be the next most limiting in severely heat treated amaranth. The FBS seems to be a promising method for popping amaranth at industrial level. PMID:2631086

  8. Predominant information quality scheme for the essential amino acids: an information-theoretical analysis.

    PubMed

    Esquivel, Rodolfo O; Molina-Espíritu, Moyocoyani; López-Rosa, Sheila; Soriano-Correa, Catalina; Barrientos-Salcedo, Carolina; Kohout, Miroslav; Dehesa, Jesús S

    2015-08-24

    In this work we undertake a pioneer information-theoretical analysis of 18 selected amino acids extracted from a natural protein, bacteriorhodopsin (1C3W). The conformational structures of each amino acid are analyzed by use of various quantum chemistry methodologies at high levels of theory: HF, M062X and CISD(Full). The Shannon entropy, Fisher information and disequilibrium are determined to grasp the spatial spreading features of delocalizability, order and uniformity of the optimized structures. These three entropic measures uniquely characterize all amino acids through a predominant information-theoretic quality scheme (PIQS), which gathers all chemical families by means of three major spreading features: delocalization, narrowness and uniformity. This scheme recognizes four major chemical families: aliphatic (delocalized), aromatic (delocalized), electro-attractive (narrowed) and tiny (uniform). All chemical families recognized by the existing energy-based classifications are embraced by this entropic scheme. Finally, novel chemical patterns are shown in the information planes associated with the PIQS entropic measures. PMID:26175003

  9. Control of Aeromonas on minimally processed vegetables by decontamination with lactic acid, chlorinated water, or thyme essential oil solution.

    PubMed

    Uyttendaele, M; Neyts, K; Vanderswalmen, H; Notebaert, E; Debevere, J

    2004-02-01

    Aeromonas is an opportunistic pathogen, which, although in low numbers, may be present on minimally processed vegetables. Although the intrinsic and extrinsic factors of minimally processed prepacked vegetable mixes are not inhibitory to the growth of Aeromonas species, multiplication to high numbers during processing and storage of naturally contaminated grated carrots, mixed lettuce, and chopped bell peppers was not observed. Aeromonas was shown to be resistant towards chlorination of water, but was susceptible to 1% and 2% lactic acid and 0.5% and 1.0% thyme essential oil treatment, although the latter provoked adverse sensory properties when applied for decontamination of chopped bell peppers. Integration of a decontamination step with 2% lactic acid in the processing line of grated carrots was shown to have the potential to control the overall microbial quality of the grated carrots and was particularly effective towards Aeromonas. PMID:14751681

  10. A simple estimation of ideal profile of essential amino acids and metabolizable energy for growing Japanese quail.

    PubMed

    Mehri, M; Ghazaghi, M; Bagherzadeh-Kasmani, F; Rokouei, M

    2016-08-01

    An experiment was conducted to determine apparent metabolizable energy (AME) and amino acid requirements of growing Japanese quail based on ideal protein concept using artificial neural network and desirability function (D-ANN). Seven-day-old quail chicks were assigned to nine experimental diets based on central composite design (CCD) containing five levels of AME (2809-3091 kcal/kg) and CP (19-24.8% of diet). The ratio of lysine (Lys) to CP was set at 0.053 among all treatments, and remaining essential amino acids (EAA) were adjusted to Lys. The experimental data of CCD were fitted to D-ANN model to compute the optimal values for independent variables. The optimal values of inputs including AME, CP, digestible Lys (dLys), methionine (dMet), total sulphur amino acids (dTSAA), threonine (dThr), tryptophan (dTrp), isoleucine (dIle), valine (dVal) and arginine (dArg) for maximizing gain and minimizing feed conversion ratio were estimated at 2865 kcal/kg, 25, 1.32, 0.55, 0.88, 0.84, 0.20, 0.75, 1.04 and 1.45% of diet, respectively, with D (desirability function) = 0.94. The corresponding optimal amounts of amino acids based on total amino acids were 1.42, 0.59, 0.95, 0.90, 0.22, 0.81, 1.12 and 1.56% of diet respectively. The ideal pattern of essential amino acids to Lys was as follows: dMet: dLys = 0.42, dTSAA: dLys = 0.67, dThr: dLys = 0.64, dTrp: dLys = 0.15, dIle: dLys = 0.57, dVal: dLys = 0.79 and dArg: dLys = 1.09. The results of this study showed that amino acid requirements of modern quails might be higher than those reported by NRC. PMID:26671312

  11. Amelioration of both early and late radiation-induced damage to pig skin by essential fatty acids

    SciTech Connect

    Hopewell, J.W.; Van den Aardweg, G.J.M.J.; Morris, G.M.

    1994-12-01

    To evaluate the possible role of essential fatty acids, specifically gamma-linolenic and eicosapentaenoic acid, in the amelioration of early and late radiation damage to the skin. Skin sites on the flank of 22-25 kg female large white pigs were irradiated with either single or fractionated doses (20 F/28 days) of {beta}-rays from 22.5 mm diameter {sup 90}Sr/{sup 90}Y plaques at a dose rate of {approximately}3 Gy/min. Essential fatty acids were administered orally in the form of two {open_quotes}active{close_quotes} oils, So-1100 and So-5407, which contained gamma-linolenic acid and a mixture of that oil with eicosapentaenoic acid, respectively. Oils (1.5-6.0 ml) were given daily for 4 weeks prior, both 4 weeks prior and 10-16 weeks after, or in the case of one single dose study, just for 10 weeks after irradiation. Control animals received a {open_quotes}placebo{close_quotes} oil, So-1129, containing no gamma linolenic acid or eicosapentaenoic acid over similar time scales before and after irradiation. Acute and late skin reactions were assessed visually and the dose-related incidence of a specific reaction used to compare the effects of different treatment schedules. A reduction in the severity of both the early and late radiation reactions in the skin was only observed when {open_quotes}active{close_quotes} oils were given over the time course of the expression of radiation damage. Prior treatment with oils did not modify the radiation reaction. A 3.0 ml daily dose of either So-1100 or So-5407 given prior to, but also after irradiation with single and fractionated doses of {beta}-rays produced the most significant modification to the radiation reactions, effects consistent with dose modification factors between 1.06-1.24 for the acute reactions of bright red erythema and/or moist desquamation, and of 1.14-1.35 for the late reactions of dusky/mauve erythema and dermal necrosis. 38 refs., 5 tabs.

  12. Relationships between circulating plasma concentrations and duodenal flows of essential amino acids in lactating dairy cows.

    PubMed

    Patton, R A; Hristov, A N; Parys, C; Lapierre, H

    2015-07-01

    The objective of this study was to better define essential AA (EAA) requirements in lactating dairy cows through examination of the relationship between plasma essential AA concentration (p[EAA]) and predicted duodenal flow of essential AA (EAAduo). Our hypothesis was that at a given level of milk protein output, p[EAA] would remain steady in response to increasing EAAduo until the EAA requirement was met, at which point p[EAA] would increase rapidly in response to greater duodenal flow of EAA until p[EAA] reached a plateau as other body processes degraded excess EAA to avoid toxicity. Thus, the requirement of each EAA would be fulfilled when p[EAA] increased rapidly. To investigate this hypothesis, we compiled a literature database that included 102 studies with 420 treatment means that reported p[EAA], dietary nutrient content, body weight, and milk production. A second database was produced to validate relationships developed in the first database and included 32 studies with 98 treatment means. All relationships were evaluated as regression equations with study as a random factor. Breed, days in milk, body weight, and milk protein production had no effect on the plasma concentration of any EAA. Other than metabolizable protein supply, nutritional content of the rations did not affect p[EAA]. Only p[Arg] was affected by parity, with primiparous cows having higher concentrations of Arg than older cows. No break points in the relationship between p[EAA] versus EAAduo were detected as either steep increases or plateaus. Plasma Arg, Ile, Lys, Thr, and Val concentrations were best associated with their respective EAAduo as quadratic equations, whereas His, Leu, Met, and Phe were associated only linearly. Adding a quadratic term improved the adjusted R(2) or decreased the root mean square error marginally (<2.0%). Thus, we conclude that the main effect of EAAduo on p[EAA] is linear. Abomasal or duodenal infusions of Met, Lys, His, Lys+Met, and casein revealed that Met

  13. Forensic applications of sodium rhodizonate and hydrochloric acid: a new histological technique for detection of gunshot residues.

    PubMed

    Andreola, Salvatore; Gentile, Guendalina; Battistini, Alessio; Cattaneo, Cristina; Zoja, Riccardo

    2011-05-01

    Demonstration of the presence of lead residues deriving from gunshot in skin and underlying tissues is essential for the correct forensic analysis of numerous legal cases. Optical microscopy remains the fastest, cheapest diagnostic technique, even though its sensitivity and specificity are poor because of the scarce quantity of histological tissue that can be examined and possible environmental lead pollution. To confirm the presence of lead from gunshot residues, we applied to histological sections of human skin a technique proposed by Owens and George in 1991 for macroscopic detection of lead on the clothing of shooting victims, involving a reaction with sodium rhodizonate and subsequent confirmation by color change on application of HCl. Our results demonstrate the technical possibility of using this macroscopic technique even on histological samples and support the need for further studies on a larger series of cases correlated with the type of ammunition and firing distance. PMID:21521219

  14. Intake of essential fatty acids in Indonesian children: secondary analysis of data from a nationally representative survey.

    PubMed

    Neufingerl, Nicole; Djuwita, Ratna; Otten-Hofman, Anke; Nurdiani, Reisi; Garczarek, Ursula; Sulaeman, Ahmad; Zock, Peter L; Eilander, Ans

    2016-02-28

    Essential fatty acids (EFA) such as α-linolenic acid (ALA) and linoleic acid (LA) are needed for healthy growth and development of children. Worldwide, reliable intake data of EFA are often lacking. The objective of this study was to investigate dietary intake of EFA in Indonesian children. Dietary intake data of 4-12-year-old children (n 45,821) from a nationally representative Indonesian survey were used to estimate median intake and distribution of population fatty acid intake. Missing data on individual fatty acids in the Indonesian food composition table were complemented through chemical analyses of national representative food samples and imputation of data from the US nutrient database. Nutrient adequacy ratios were calculated as a percentage of FAO/WHO intake recommendations. The medians of total fat intake of the children was 26·7 (10th-90th percentile 11·2-40·0) percentage of total daily energy (%E). Intakes of fatty acids were 4·05 (10th-90th percentile 1·83-7·22) %E for total PUFA, 3·36 (10th-90th percentile 1·14-6·29) %E for LA and 0·20 (10th-90th percentile 0·07-0·66) %E for ALA. Median intake of PUFA was 67 % and that of ALA 40 % of the minimum amounts recommended by FAO/WHO. These data indicate that a majority of Indonesian children has intakes of PUFA and specifically ALA that are lower than recommended intake levels. Total fat and LA intakes may be suboptimal for a smaller yet considerable proportion of children. Public health initiatives should provide practical guidelines to promote consumption of PUFA-rich foods. PMID:26824732

  15. Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway.

    PubMed

    Cronan, John E

    2016-06-01

    Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism. PMID:27074917

  16. Maintenance of essential amino acid synthesis pathways in the Blattabacterium cuenoti symbiont of a wood-feeding cockroach

    PubMed Central

    Tokuda, Gaku; Elbourne, Liam D. H.; Kinjo, Yukihiro; Saitoh, Seikoh; Sabree, Zakee; Hojo, Masaru; Yamada, Akinori; Hayashi, Yoshinobu; Shigenobu, Shuji; Bandi, Claudio; Paulsen, Ian T.; Watanabe, Hirofumi; Lo, Nathan

    2013-01-01

    In addition to harbouring intestinal symbionts, some animal species also possess intracellular symbiotic microbes. The relative contributions of gut-resident and intracellular symbionts to host metabolism, and how they coevolve are not well understood. Cockroaches and the termite Mastotermes darwiniensis present a unique opportunity to examine the evolution of spatially separated symbionts, as they harbour gut symbionts and the intracellular symbiont Blattabacterium cuenoti. The genomes of B. cuenoti from M. darwiniensis and the social wood-feeding cockroach Cryptocercus punctulatus are each missing most of the pathways for the synthesis of essential amino acids found in the genomes of relatives from non-wood-feeding hosts. Hypotheses to explain this pathway degradation include: (i) feeding on microbes present in rotting wood by ancestral hosts; (ii) the evolution of high-fidelity transfer of gut microbes via social behaviour. To test these hypotheses, we sequenced the B. cuenoti genome of a third wood-feeding species, the phylogenetically distant and non-social Panesthia angustipennis. We show that host wood-feeding does not necessarily lead to degradation of essential amino acid synthesis pathways in B. cuenoti, and argue that ancestral high-fidelity transfer of gut microbes best explains their loss in strains from M. darwiniensis and C. punctulatus. PMID:23515978

  17. Maintenance of essential amino acid synthesis pathways in the Blattabacterium cuenoti symbiont of a wood-feeding cockroach.

    PubMed

    Tokuda, Gaku; Elbourne, Liam D H; Kinjo, Yukihiro; Saitoh, Seikoh; Sabree, Zakee; Hojo, Masaru; Yamada, Akinori; Hayashi, Yoshinobu; Shigenobu, Shuji; Bandi, Claudio; Paulsen, Ian T; Watanabe, Hirofumi; Lo, Nathan

    2013-06-23

    In addition to harbouring intestinal symbionts, some animal species also possess intracellular symbiotic microbes. The relative contributions of gut-resident and intracellular symbionts to host metabolism, and how they coevolve are not well understood. Cockroaches and the termite Mastotermes darwiniensis present a unique opportunity to examine the evolution of spatially separated symbionts, as they harbour gut symbionts and the intracellular symbiont Blattabacterium cuenoti. The genomes of B. cuenoti from M. darwiniensis and the social wood-feeding cockroach Cryptocercus punctulatus are each missing most of the pathways for the synthesis of essential amino acids found in the genomes of relatives from non-wood-feeding hosts. Hypotheses to explain this pathway degradation include: (i) feeding on microbes present in rotting wood by ancestral hosts; (ii) the evolution of high-fidelity transfer of gut microbes via social behaviour. To test these hypotheses, we sequenced the B. cuenoti genome of a third wood-feeding species, the phylogenetically distant and non-social Panesthia angustipennis. We show that host wood-feeding does not necessarily lead to degradation of essential amino acid synthesis pathways in B. cuenoti, and argue that ancestral high-fidelity transfer of gut microbes best explains their loss in strains from M. darwiniensis and C. punctulatus. PMID:23515978

  18. Urea, glycolic acid, and glycerol in an organic residue produced by ultraviolet irradiation of interstellar/pre-cometary ice analogs.

    PubMed

    Nuevo, Michel; Bredehöft, Jan Hendrik; Meierhenrich, Uwe J; d'Hendecourt, Louis; Thiemann, Wolfram H-P

    2010-03-01

    More than 50 stable organic molecules have been detected in the interstellar medium (ISM), from ground-based and onboard-satellite astronomical observations, in the gas and solid phases. Some of these organics may be prebiotic compounds that were delivered to early Earth by comets and meteorites and may have triggered the first chemical reactions involved in the origin of life. Ultraviolet irradiation of ices simulating photoprocesses of cold solid matter in astrophysical environments have shown that photochemistry can lead to the formation of amino acids and related compounds. In this work, we experimentally searched for other organic molecules of prebiotic interest, namely, oxidized acid labile compounds. In a setup that simulates conditions relevant to the ISM and Solar System icy bodies such as comets, a condensed CH(3)OH:NH(3) = 1:1 ice mixture was UV irradiated at approximately 80 K. The molecular constituents of the nonvolatile organic residue that remained at room temperature were separated by capillary gas chromatography and identified by mass spectrometry. Urea, glycolic acid, and glycerol were detected in this residue, as well as hydroxyacetamide, glycerolic acid, and glycerol amide. These organics are interesting target molecules to be searched for in space. Finally, tentative mechanisms of formation for these compounds under interstellar/pre-cometary conditions are proposed. PMID:20402585

  19. Fatty acid carbon is essential for dNTP synthesis in endothelial cells

    PubMed Central

    Missiaen, Rindert; Queiroz, Karla CS; Borgers, Gitte; Elia, Ilaria; Zecchin, Annalisa; Cantelmo, Anna Rita; Christen, Stefan; Goveia, Jermaine; Heggermont, Ward; Goddé, Lucica; Vinckier, Stefan; Van Veldhoven, Paul P.; Eelen, Guy; Schoonjans, Luc; Gerhardt, Holger; Dewerchin, Mieke; Baes, Myriam; De Bock, Katrien; Ghesquière, Bart; Lunt, Sophia Y.; Fendt, Sarah-Maria; Carmeliet, Peter

    2015-01-01

    The metabolism of endothelial cells (ECs) during vessel sprouting remains poorly studied. Here, we report that endothelial loss of CPT1a, a rate-limiting enzyme of fatty acid oxidation (FAO), caused vascular sprouting defects due to impaired proliferation, not migration of ECs. Reduction of FAO in ECs did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labeling studies in control ECs showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1a silencing reduced these processes and depleted EC stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1a-silenced ECs. Finally, CPT1 blockade inhibited pathological ocular angiogenesis, suggesting a novel strategy for blocking angiogenesis. PMID:25830893

  20. Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol-Acid Reductoisomerase.

    PubMed

    Tadrowski, Sonya; Pedroso, Marcelo M; Sieber, Volker; Larrabee, James A; Guddat, Luke W; Schenk, Gerhard

    2016-05-23

    Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions. PMID:27136273

  1. The transcriptional coactivator PGC-1α is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

    PubMed Central

    Lehman, John J.; Boudina, Sihem; Banke, Natasha Hausler; Sambandam, Nandakumar; Han, Xianlin; Young, Deanna M.; Leone, Teresa C.; Gross, Richard W.; Lewandowski, E. Douglas; Abel, E. Dale; Kelly, Daniel P.

    2008-01-01

    High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1α (PGC-1α−/− mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1α is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1α−/− mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1α−/− mice compared with PGC-1α+/+ controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1α−/− mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1α−/− fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1α−/− mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. 13C NMR revealed that hearts from PGC-1α−/− mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during β-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1α−/− mice relative to PGC-1α+/+ following a fast. Overall, these results demonstrate that PGC-1α is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP

  2. Effect of Coriandrum sativum hydroalcoholic extract and its essential oil on acetic acid- induced acute colitis in rats

    PubMed Central

    Heidari, Bahareh; Sajjadi, Seyed Ebrahim; Minaiyan, Mohsen

    2016-01-01

    Objective: The aim of this study was to determine the protective effects of Coriandrum sativum on acetic acid-inducedcolitis in rats. C. sativum (Coriander) has long been used in Iranian traditional medicine and its use as an anti-inflammatory agent is still common in some herbal formulations. Materials and Methods: Colitis was induced by intra-rectal administration of 2ml acetic acid 4% in fasted male Wistar rats. Treatment was carried out using three increasing doses of extract (250, 500, 1000 mg/kg) and essential oil (0.25, 0.5, 1 ml/kg) of coriander started 2 h before colitis induction and continued for a five-day period. Colon biopsies were taken for weighting, macroscopic scoring of injured tissue, histopathological examination and measuring myeloperoxidase (MPO) activity. Results: Colon weight was decreased in the groups treated with extract (500 and 1000 mg/kg) and essential oil (0.5 ml/kg) compared to the control group. Regarding MPO levels, ulcer severity and area as well as the total colitis index, same results indicating meaningful alleviation of colitis was achieved after treatment with oral extract and essential oil. Conclusion: Since the present experiment was made by oral fractions of coriander thus the resulting effects could be due to both the absorption of the active ingredients and/or the effect of non-absorbable materials on colitis after reaching the colon. In this regard, we propose more toxicological and clinical experiments to warranty its beneficial application in human inflammatory bowel diseases. PMID:27222834

  3. Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

    PubMed Central

    Rokic, Milos B.; Stojilkovic, Stanko S.; Vavra, Vojtech; Kuzyk, Pavlo; Tvrdonova, Vendula; Zemkova, Hana

    2013-01-01

    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. PMID:23555667

  4. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    PubMed

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs. PMID:25107664

  5. Influence of shear force on floc properties and residual aluminum in humic acid treatment by nano-Al₁₃.

    PubMed

    Xu, Weiying; Gao, Baoyu; Du, Bin; Xu, Zhenghe; Zhang, Yongfang; Wei, Dong

    2014-04-30

    The impacts of various shear forces on floc sizes and structures in humic acid coagulations by polyaluminum chloride (PACl) and nano-Al13 were comparatively studied in this paper. The dynamic floc size was monitored by use of a laser diffraction particle sizing device. The floc structure was evaluated in terms of fractal dimension, analyzed by small-angle laser light scattering (SALLS). The effect of increased shear rate on residual Al of the coagulation effluents was then analyzed on the basis of different floc characteristics generated under various shear conditions. The results showed that floc size decreased with the increasing shear rate for both Al13 and PACl. Besides, floc strength and re-formation ability were also weakened by the enhanced shear force. Al13 resulted in small, strong and better recoverable flocs than PACl and moreover, in the shear range of 100-300 revolution per minute (rpm) (G=40.7-178.3s(-1)), the characteristics of HA-Al13 flocs displayed smaller scale changes than those of HA-PACl flocs. The results of residual Al measurements proved that with shear increased, the residual Al increased continuously but Al13 presented less sensitivity to the varying shear forces. PACl contributed higher residual Al than Al13 under the same shear condition. PMID:24583809

  6. Diversity in the ability of cultured cells to elongate and desaturate essential (n-6 and n-3) fatty acids.

    PubMed

    Grammatikos, S I; Subbaiah, P V; Victor, T A; Miller, W M

    1994-11-30

    Essential fatty acids (EFAs) cannot be synthesized by mammalian cells. Once taken in with the diet, they can undergo desaturations/saturations and chain elongations/shortenings to yield a variety of polyunsaturated fatty acids of the same family. Cells in vitro from a variety of tissues are capable of processing EFAs to varying extents. Conversion of the parent EFAs, linoleic (LA, n-6) and alpha-linolenic (LNA, n-3) acids, to the 20-carbon polyunsaturated fatty acids, arachidonic (AA, n-6) and eicosapentanoic (EPA, n-3), requires chain elongation and delta 6 and delta 5 desaturations. AA and EPA are required by many tissues for optimal biological function and are precursors of biologically active eicosanoid hormones. All cultured cells are able to elongate exogenous LA and LNA, and most can perform delta 5 desaturation, so delta 6 desaturation is the limiting step in AA and EPA production. Longer fatty acids that have more double bonds than AA or EPA are less frequently produced due to a deficiency in delta 4 desaturating ability. The process of retroconversion (chain shortening) is less extensively studied, but evidence from a variety of cells suggests that this type of metabolic conversion is normally active. The example of MCF-7 (human breast cancer cell line) and MCF-10A cells (human noncancerous breast cell line) is discussed in order to emphasize the diversity in EFA processing ability of cultured cells. Under identical culture conditions, MCF-10A cells perform extensive desaturations, elongations, and retroconversions, whereas MCF-7 cells can only elongate and retroconvert exogenous EFAs. Given the great diversity in the ability of cultured cells to process EFAs, no conclusions can be drawn regarding the mechanisms responsible for the effects of exogenous EFAs on a particular cell until that cell's EFA processing patterns have been evaluated. PMID:7832535

  7. Identification of Significant Amino Acids in Multiple Transmembrane Domains of Human Transient Receptor Potential Ankyrin 1 (TRPA1) for Activation by Eudesmol, an Oxygenized Sesquiterpene in Hop Essential Oil

    PubMed Central

    Ohara, Kazuaki; Fukuda, Takafumi; Okada, Hiroyuki; Kitao, Sayoko; Ishida, Yuko; Kato, Kyoko; Takahashi, Chika; Katayama, Mikio; Uchida, Kunitoshi; Tominaga, Makoto

    2015-01-01

    Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca2+ measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03–0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by β-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals. PMID:25525269

  8. Optimizing dietary lipid use to improve essential fatty acid status and reproductive performance of the modern lactating sow: a review.

    PubMed

    Rosero, David S; Boyd, R Dean; Odle, Jack; van Heugten, Eric

    2016-01-01

    Dietary lipid supplementation benefits the prolific and high-producing modern lactating sow. A comprehensive review of recent studies showed that lipid supplementation increases average daily energy intake, which is partitioned for lactation as indicated by greater milk fat output and improved litter growth rate. Recent compelling findings showed that addition of particular lipids during lactation improved the subsequent reproductive outcome of sows. Such benefits were related to the level of dietary essential fatty acids (EFA, linoleic acid, C18:2n-6; and α-linolenic acid, C18:3n-3) during lactation. Lactation diets without supplemental EFA resulted in a pronounced negative balance (intake minus milk output) of linoleic (-25.49 g/d) and α-linolenic acid (-2.75 g/d); which compromised sow fertility (farrowing rate < 75 % and culling rates > 25 % of weaned sows). This phenomenon seems to be increasingly important with advancing sow age because of a progressive reduction of body EFA pool over successive lactations. The net effect of supplemental EFA during lactation was to create a positive EFA balance, which improved the subsequent reproduction of sows. Adequate linoleic acid intake improved the proportion of sows that farrowed in the subsequent cycle (Farrowing rate (%) = [(-1.5 × 10(-3) × linoleic acid intake (g/d)(2)) + (0.53 × linoleic acid intake (g/d)) + (45.2)]; quadratic P = 0.002, R(2) = 0.997, RMSE = 0.031). In addition, increasing linoleic acid intake increased the number of pigs born in the subsequent cycle (total pigs born (n) = [(9.4 × 10(-5) × linoleic acid intake (g/d)(2)) + (0.04 × linoleic acid intake (g/d)) + (10.94)]; quadratic P = 0.002, R(2) = 0.997, RMSE = 0.031). Supplemental α-linolenic acid resulted in a rapid return to estrus (sows bred: sows weaned = 94.2 %; wean-to-estrus interval = 4.0 d) and achieved a high retention of pregnancy (sows

  9. Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates.

    PubMed

    Geng, Meimei; Li, Tiejun; Kong, Xiangfeng; Song, Xiaoyan; Chu, Wuying; Huang, Ruilin; Yin, Yulong; Wu, Guoyao

    2011-05-01

    The objective of this study was to determine developmental changes in mRNA and protein levels for N-acetylglutamate synthase (NAGS; a key enzyme in synthesis of citrulline and arginine from glutamine/glutamate and proline) in the small intestine of suckling piglets. The porcine NAGS gene was cloned using the real-time polymerase-chain reaction (RT-PCR) method. The porcine NAGS gene encoded 368 amino acid residues and had a high degree of sequence similarity to the "conserved domain" of human and mouse NAGS genes. The porcine NAGS gene was expressed in E. coli BL21 and a polyclonal antibody against the porcine NAGS protein was developed. Real-time RT-PCR and western-blot analyses were performed to quantify NAGS mRNA and protein, respectively, in the jejunum and ileum of 1- to 28-day-old pigs. Results indicated that intestinal NAGS mRNA levels were lower in 7- to 28-day-old than in 1-day-old pigs. Immunochemical analysis revealed that NAGS protein was localized in enterocytes of the gut. Notably, intestinal NAGS protein abundance declined progressively during the 28-day suckling period. The postnatal decrease in NAGS protein levels was consistent with the previous report of reduced NAGS enzymatic activity as well as reduced synthesis of citrulline and arginine in the small intestine of 7- to 28-day-old pigs. Collectively, these results suggest that intestinal NAGS expression is regulated primarily at the post-transcriptional level. The findings also provide a new molecular basis to explain that endogenous synthesis of arginine is impaired in sow-reared piglets and arginine is a nutritionally essential amino acid for the neonates. PMID:20931344

  10. Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages

    NASA Astrophysics Data System (ADS)

    Li, Jinxi; Shefcheck, Kevin; Callahan, John; Fenselau, Catherine

    2008-12-01

    This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.

  11. Slr2019, lipid A transporter homolog, is essential for acidic tolerance in Synechocystis sp. PCC6803.

    PubMed

    Matsuhashi, Ayumi; Tahara, Hiroko; Ito, Yutaro; Uchiyama, Junji; Ogawa, Satoru; Ohta, Hisataka

    2015-08-01

    Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41 %. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60 %. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56 %. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for

  12. Influence of sex and gonadal hormones on rat-liver and carcass lipids during the development of an essential fatty acid deficiency

    PubMed Central

    Ostwald, Rosemarie; Bouchard, Pauline; Miljanich, P.; Lyman, R. L.

    1965-01-01

    1. Groups of intact male and female rats and castrated rats injected with oestradiol or testosterone were given a diet containing hydrogenated coconut oil for 9 weeks, and at intervals the amounts and fatty acid compositions of the carcass and liver lipids were determined. 2. Male rats grew faster and larger, and exhibited typical external essential fatty acid deficiency symptoms sooner than did females. Testosterone-treated castrated male rats were similar to males, and oestradiol-injected castrated male rats resembled females. 3. Intact females maintained a higher linoleic acid concentration in their carcass than did males. Total amounts of carcass linoleic acid remained similar for all groups, only 200mg. being removed in 9 weeks regardless of body size. 4. The amounts of total cholesteryl esters were independent of liver size. They were higher in males and testosterone-treated castrated male rats than in females and oestrogen-treated castrated male rats. 5. Phospholipids represented about 80% of the liver lipids. The total amounts of the phospholipid linoleic acid and arachidonic acid were similar for all groups regardless of liver size, and were not affected appreciably by the deficiency. Females and oestrogen-treated castrated male rats maintained a higher proportion of phospholipid arachidonic acid for longer periods than did their male counterparts. Both the total amounts and the proportions of eicosatrienoic acid and palmitic acid were higher in males than in females. 6. Supplementation of the essential fatty acid-deficient diet with linoleic acid caused a rapid loss of eicosatrienoic acid and palmitic acid with a concomitant increase in stearic acid and arachidonic acid. 7. There were no obvious differences in the way that the essential fatty acids were metabolized or mobilized from adipose tissue of male or female rats during essential fatty acid deficiency. 8. The results indicated that the greater growth rate of the male rats caused them to require and

  13. Early development of essential fatty acid deficiency in rats: Fat-free vs. hydrogenated coconut oil diet

    PubMed Central

    Ling, Pei-Ra; De Leon, Charlotte E.; Le, Hau; Puder, Mark; Bistrian, Bruce R.

    2011-01-01

    This study examined the effects of feeding an essential fatty acid deficient (EFAD) diet either without fat or with added hydrogenated coconut oil (HCO) on fatty acid profiles in rats. Both diets induced equivalent biochemical evidence of EFAD reflected by the triene/tetraene ratio in plasma phospholipids within 2 weeks. However, the HCO diet led to larger increases of 16:1n7 and 18:1n9 in muscle but smaller increases in fat tissue and plasma triglycerides than the fat-free diet, suggesting greater increases in hepatic de novo lipogenesis with the latter. In addition, the HCO diet led to larger decreases of some 18:3n3 metabolites, particularly 22:6n3, in muscle, fat and brain tissues than the fat-free diet, presumably related to lesser stimulation of elongation and desaturation. Thus, these secondary effects of an EFAD diet on fatty acid metabolism can be modified by the saturated fat in the diet while the primary impact of both diets on development of EFAD is unaffected. PMID:20675109

  14. Serum uric acid level in newly diagnosed essential hypertension in a Nepalese population: A hospital based cross sectional study

    PubMed Central

    Poudel, Bibek; Yadav, Binod Kumar; Kumar, Arun; Jha, Bharat; Raut, Kanak Bahadur

    2014-01-01

    Objective To develop the missing link between hyperuricemia and hypertension. Methods The study was conducted in Department of Biochemistry in collaboration with Nephrology Unit of Internal Medicine Department. Hypertension was defined according to blood pressure readings by definitions of the Seventh Report of the Joint National Committee. Totally 205 newly diagnosed and untreated essential hypertensive cases and age-sex matched normotensive controls were enrolled in the study. The potential confounding factors of hyperuricemia and hypertension in both cases and controls were controlled. Uric acid levels in all participants were analyzed. Results Renal function between newly diagnosed hypertensive cases and normotensive healthy controls were adjusted. The mean serum uric acid observed in newly diagnosed hypertensive cases and in normotensive healthy controls were (290.05±87.05) µmol/L and (245.24±99.38) µmol/L respectively. A total of 59 (28.8%) participants of cases and 28 (13.7%) participants of controls had hyperuricemia (odds ratio 2.555 (95% CI: 1.549-4.213), P<0.001). Conclusions The mean serum uric acid levels and number of hyperuricemic subjects were found to be significantly higher in cases when compared to controls. PMID:24144132

  15. Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin.

    PubMed

    Cervia, Davide; Assi, Emma; De Palma, Clara; Giovarelli, Matteo; Bizzozero, Laura; Pambianco, Sarah; Di Renzo, Ilaria; Zecchini, Silvia; Moscheni, Claudia; Vantaggiato, Chiara; Procacci, Patrizia; Clementi, Emilio; Perrotta, Cristiana

    2016-05-01

    The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy. PMID:27107419

  16. C-terminal amino acids are essential for human heat shock protein 70 dimerization.

    PubMed

    Marcion, Guillaume; Seigneuric, Renaud; Chavanne, Evelyne; Artur, Yves; Briand, Loïc; Hadi, Tarik; Gobbo, Jessica; Garrido, Carmen; Neiers, Fabrice

    2015-01-01

    The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity. PMID:25030382

  17. The fatty acid elongase Bond is essential for Drosophila sex pheromone synthesis and male fertility

    PubMed Central

    Ng, Wan Chin; Chin, Jacqueline S. R.; Tan, Kah Junn; Yew, Joanne Y.

    2015-01-01

    Insects use a spectacular variety of chemical signals to guide their social behaviours. How such chemical diversity arises is a long-standing problem in evolutionary biology. Here we describe the contribution of the fatty acid elongase Bond to both pheromone diversity and male fertility in Drosophila. Genetic manipulation and mass spectrometry analysis reveal that the loss of bond eliminates the male sex pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Unexpectedly, silencing bond expression severely suppresses male fertility and the fertility of conspecific rivals. These deficits are rescued on ectopic expression of bond in the male reproductive system. A comparative analysis across six Drosophila species shows that the gain of a novel transcription initiation site is correlated with bond expression in the ejaculatory bulb, a primary site of male pheromone production. Taken together, these results indicate that modification of cis-regulatory elements and subsequent changes in gene expression pattern is one mechanism by which pheromone diversity arises. PMID:26369287

  18. Cutting Edge: Retinoic Acid Signaling in B Cells Is Essential for Oral Immunization and Microflora Composition.

    PubMed

    Pantazi, Eirini; Marks, Ellen; Stolarczyk, Emilie; Lycke, Nils; Noelle, Randolph J; Elgueta, Raul

    2015-08-15

    Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA(+) Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition. PMID:26163586

  19. Identification of functionally important amino acid residues in the mitochondria targeting sequence of Hepatitis B virus X protein

    SciTech Connect

    Li, Sai Kam; Ho, Sai Fan; Tsui, Kwok Wing; Fung, Kwok Pui; Waye, M.Y. Mary

    2008-11-10

    Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated.

  20. Supplementation of essential fatty acids to Holstein calves during late uterine life and first month of life alters hepatic fatty acid profile and gene expression.

    PubMed

    Garcia, M; Greco, L F; Lock, A L; Block, E; Santos, J E P; Thatcher, W W; Staples, C R

    2016-09-01

    Linoleic acid is an essential dietary fatty acid (FA). However, how the supplementation of linoleic acid during uterine and early life may modify the FA profile and transcriptome regulation of the liver, and performance of preweaned dairy calves is unknown. Our objective was to evaluate the effect of supplementation of essential FA to Holstein calves during late uterine and early life on their hepatic FA profile and global gene expression at 30 d of age. During the last 8 wk of pregnancy, Holstein cattle (n=96) were fed either no fat supplement (control), a saturated FA supplement enriched with C18:0, or an unsaturated FA supplement enriched with linoleic acid. Male calves (n=40) born from these dams were fed a milk replacer (MR) with either low (LLA) or high linoleic acid (HLA) concentration as the sole feedstuff during the first 30 d. Liver biopsy was performed at 30 d of age, and microarray analysis was performed on 18 liver samples. Total concentration of FA in liver were greater in calves fed LLA compared with those fed HLA MR (8.2 vs. 7.1%), but plasma concentrations of total FA did not differ due to MR diets. The FA profiles of plasma and liver of calves were affected differently by the prepartum diets. Specifically, the FA profile in liver was affected moderately by the feeding of fat prepartum, but the profiles did not differ due to the type of FA fed prepartum. The type of MR fed during the first 30 d of life had major effects on both plasma and liver FA profiles, resembling the type of fat fed. Plasma and liver of calves fed LLA MR had greater percentage of medium-chain FA (C12:0 and C14:0), whereas plasma and liver from calves fed HLA MR had greater percentages of linoleic and α-linolenic acids. Dams fed fat or a specific type of FA modified the expression of some genes in liver of calves, particularly those genes involved in biological functions and pathways related to upregulation of lipid metabolism and downregulation of inflammatory responses

  1. Toxin acidic residue evolutionary function-guided design of de novo peptide drugs for the immunotherapeutic target, the Kv1.3 channel

    PubMed Central

    Chen, Zongyun; Hu, Youtian; Hong, Jing; Hu, Jun; Yang, Weishan; Xiang, Fang; Yang, Fan; Xie, Zili; Cao, Zhijian; Li, Wenxin; Lin, Donghai; Wu, Yingliang

    2015-01-01

    During the long-term evolution of animal toxins acting on potassium channels, the acidic residues can orientate the toxin binding interfaces by adjusting the molecular polarity. Based on the evolutionary function of toxin acidic residues, de novo peptide drugs with distinct binding interfaces were designed for the immunotherapeutic target, the Kv1.3 channel. Using a natural basic toxin, BmKTX, as a template, which contains 2 acidic residues (Asp19 and Asp33), we engineered two new peptides BmKTX-19 with 1 acidic residue (Asp33), and BmKTX-196 with 2 acidic residues (Asp6 and Asp33) through only adjusting acidic residue distribution for reorientation of BmKTX binding interface. Pharmacological experiments indicated that BmKTX-19 and BmKTX-196 peptides were specific inhibitors of the Kv1.3 channel and effectively suppressed cytokine secretion. In addition to the structural similarity between the designed and native peptides, both experimental alanine-scanning mutagenesis and computational simulation further indicated that the binding interface of wild-type BmKTX was successfully reoriented in BmKTX-19 and BmKTX-196, which adopted distinct toxin surfaces as binding interfaces. Together, these findings indicate not only the promising prospect of BmKTX-19 and BmKTX-196 as drug candidates but also the desirable feasibility of the evolution-guided peptide drug design for discovering numerous peptide drugs for the Kv1.3 channel. PMID:25955787

  2. Preparation of a modified flue gas desulphurization residue and its effect on pot sorghum growth and acidic soil amelioration.

    PubMed

    Shi, Lin; Xu, Peizhi; Xie, Kaizhi; Tang, Shuanhu; Li, Yongli

    2011-09-15

    A modified flue gas desulphurization residue (MFGDR) was prepared and its effects on sorghum growth and acidic soil amelioration were evaluated in this paper. The MFGDR was prepared by calcining a mixture of dry/semi-dry flue gas desulphurization (FGD) residue from a coal-fired power plant, sorted potash feldspar and/or limestone powder. The available nutrients from the MFGDR were determined with 4.91 wt% K(+), 1.15 wt% Mg(2+), 22.4 wt% Ca(2+), 7.01 wt% Si(4+) and 2.07 wt% SO(4)(2-)-S in 0.1 mol L(-1) citric acid solution. Its pH value was held at 9.60 displaying slightly alkaline. The results of sorghum pot growth in both red and crimson acidic soil for 30 days indicated that adding the MFGDR at a dosage of 2 g kg(-1) in total soil weight would increase the growth rate of biomass by 24.3-149% (wet weight basis) and 47.3-157% (dry weight), the stem length and thickness increase by 5.75-22.1% and 4.76-30.9% in contrast with CK treatment for two test cuttings, respectively. The effect on sorghum growth was attributed to the increase of available nutrients, the enhancement of soil pH value and the reduction of aluminum toxicity in acidic soil due to the addition of the MFGDR. The experimental results also suggested that the MFGDR could be effectively used to ameliorate the acidic soil which is widely distributed throughout the southern China. PMID:21763070

  3. Simulation of acid hydrolysis of lignocellulosic residues to fermentable sugars for bioethanol production

    NASA Astrophysics Data System (ADS)

    Sidiras, Dimitris

    2012-12-01

    The dilute acid hydrolysis of fir sawdust with sulfuric acid was undertaken in a batch reactor system (autoclave). The experimental data and reaction kinetic analysis indicate that this is a potential process for cellulose and hemicelluloses hydrolysis, due to a rapid hydrolysis reaction for acid concentration 0.045 N at 160-180°C. It was found that significant sugar degradation occurred at these conditions. The optimum conditions gave a yield of 38% total fermentable sugars. The kinetics of dilute acid hydrolysis of cellulose and hemicelluloses (polysaccharides) were simulated using four pseudo-kinetic models. The reaction rate constants were calculated in each case.

  4. Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

    PubMed Central

    Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

    2001-01-01

    The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

  5. Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

    PubMed Central

    Perrin-Tricaud, Claire; Rutschmann, Christoph; Hennet, Thierry

    2011-01-01

    Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein. PMID:22216269

  6. Stabilization of emulsion and butter like products containing essential fatty acids using kalonji seeds extract and curcuminoids.

    PubMed

    Rege, Sameera A; Momin, Shamim A; Bhowmick, Dipti N; Pratap, Amit A

    2012-01-01

    Owing to the tendency of essential fatty acids (EFAs) to undergo autoxidation, their storage becomes a key problem. Generally, they are stabilized by synthetic antioxidants like TBHQ that are toxic in nature. Recently many studies were reported where these EFAs are stabilized by natural antioxidants. In the present study, curcuminoids and kalonji seeds ethanol extract (KEE) were used to stabilize these EFAs in refined sunflower oil (RSFO), water-in-oil (w/o) emulsion and butter like products (BLPs). In RSFO, though curcuminoids alone exerted pro-oxidant effect, KEE and curcuminoids showed synergistic antioxidant activity that was comparable to TBHQ. KEE exhibited good antioxidant activity in emulsions and BLPs, providing fine physical properties like slipping point, dropping point and spreadability. EFAs increased the nutritional value of BLPs and antioxidants added for their stabilization provided their medicinal benefits. PMID:22188801

  7. Amino acid residues in the Ler protein critical for derepression of the LEE5 promoter in enteropathogenic E. coli.

    PubMed

    Choi, Su-Mi; Jeong, Jae-Ho; Choy, Hyon E; Shin, Minsang

    2016-08-01

    Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region. PMID:27480636

  8. Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)-Substituted Polyoxometalates.

    PubMed

    Ly, Hong Giang T; Absillis, Gregory; Janssens, Rik; Proost, Paul; Parac-Vogt, Tatjana N

    2015-06-15

    SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site. PMID:25950869

  9. Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability.

    PubMed

    Veyron-Churlet, Romain; Guerrini, Olivier; Mourey, Lionel; Daffé, Mamadou; Zerbib, Didier

    2004-12-01

    Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions. PMID:15554959

  10. Rapid effects of essential fatty acid deficiency on growth and development parameters and transcription of key fatty acid metabolism genes in juvenile barramundi (Lates calcarifer).

    PubMed

    Salini, Michael J; Turchini, Giovanni M; Wade, Nicholas M; Glencross, Brett D

    2015-12-14

    Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, the response of juvenile barramundi (47·0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8·2% added oil was tested. The experimental test diets were either devoid of fish oil (FO), and thus with no n-3 LC-PUFA (FO FREE diet), or with a low inclusion of FO (FO LOW diet). These were compared against a control diet containing only FO (FO CTRL diet) as the added lipid source, over an 8-week period. Interim samples and measurements were taken fortnightly during the trial in order to define the aetiology of the onset and progression of EFA deficiency. After 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 weeks significant differences were detected for all performance parameters. The fish fed the FO FREE diet also had a significantly higher incidence of external abnormalities. The transcription of several genes involved in fatty acid metabolism was affected after 2 weeks of feeding, showing a rapid nutritional regulation. This experiment documents the aetiology of the onset and the progression of EFA deficiency in juvenile barramundi and demonstrates that such deficiencies can be detected within 2 weeks in juvenile fish. PMID:26411329

  11. The synergistic preservative effects of the essential oils of sweet basil (Ocimum basilicum L.) against acid-tolerant food microflora.

    PubMed

    Lachowicz, K J; Jones, G P; Briggs, D R; Bienvenu, F E; Wan, J; Wilcock, A; Coventry, M J

    1998-03-01

    Essential oils extracted by hydrodistillation from five different varieties of Ocimum basilicum L. plants (Anise, Bush, Cinnamon, Dark Opal and a commercial sample of dried basil) were examined for antimicrobial activity against a wide range of foodborne Gram-positive and -negative bacteria, yeasts and moulds by an agar well diffusion method. All five essential oils of basil showed antimicrobial activity against most of the organisms tested with the exception of Flavimonas oryzihabitans and Pseudomonas species. The inhibitory effect of Anise oil, in comparison with mixtures of the predominant components of pure linalool and methyl chavicol, against the acid-tolerant organisms, Lactobacillus curvatus and Saccharomyces cerevisiae, was examined in broth by an indirect impedance method. Synergistic effects between Anise oil, low pH (pH 4.2) and salt (5% NaCl) were determined. The antimicrobial effect of Anise oil was also assessed in a tomato juice medium by direct viable count, showing that the growth of Lact. curvatus and S. cerevisiae was completely inhibited by 0.1% and 1% Anise oil, respectively. The results of the current study indicate the need for further investigations to understand the antimicrobial effects of basil oils in the presence of other food ingredients and preservation parameters. PMID:9569711

  12. Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase

    PubMed Central

    Dellas, Nikki; Snyder, Jamie C.; Dills, Michael; Nicolay, Sheena J.; Kerchner, Keshia M.; Brumfield, Susan K.; Lawrence, C. Martin

    2015-01-01

    ABSTRACT Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives

  13. Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.

    PubMed Central

    Xu-Welliver, M; Kanugula, S; Loktionova, N A; Crone, T M; Pegg, A E

    2000-01-01

    The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this

  14. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    NASA Astrophysics Data System (ADS)

    He, Yi; Liwo, Adam; Scheraga, Harold A.

    2015-12-01

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field.

  15. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  16. Controlling fine particulate and acid mist emissions from a residual oil fired utility boiler with an EDV{trademark} system

    SciTech Connect

    Olen, K.R.; Vincent, H.B.; Jones, G.

    1995-06-01

    Florida Power & Light Company (FPL), in cooperation with the Electric Power Research Institute (EPRI) and Belco Technologies Corporation, evaluated the performance of an EDV system to remove fine particulate and acid mist from untreated flue gas from a residual oil-fired utility boiler. The cosponsored project was carried out using a full-scale EDV module in a slip stream from one of the 400 MW wall-fired boilers at FPL`s Sanford Plant. Particulate, acid gas and chemical analytical data are presented, and used to illustrate the effects of operating variables on EDV performance. EDV system efficiencies of 90% were achieved, which resulted in controlled particulate and SO{sub 3} emissions of less than 10 mg/Nm{sup 3} (0.0065 lbs/10{sup 6}Btu) and 1 ppmv, respectively.

  17. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    SciTech Connect

    He, Yi; Scheraga, Harold A.; Liwo, Adam

    2015-12-28

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field.

  18. Stabile Chlorine Isotope Study of Martian Shergottites and Nakhlites; Whole Rock and Acid Leachates and Residues

    NASA Technical Reports Server (NTRS)

    Nakamura, N.; Nyquist, L. E.; Reese, Y.; Shih, C-Y; Fujitani, T.; Okano, O.

    2011-01-01

    We have established a precise analytical technique for stable chlorine isotope measurements of tiny planetary materials by TIMS (Thermal Ionization Mass Spectrometry) [1], for which the results are basically consistent with the IRMS tech-nique (gas source mass spectrometry) [2,3,4]. We present here results for Martian shergottites and nakhlites; whole rocks, HNO3-leachates and residues, and discuss the chlorine isotope evolution of planetary Mars.

  19. Radionuclide Leaching from Residual Solids Remaining after Acid Dissolution of K East Area Sludge Composite

    SciTech Connect

    Delegard, C.H.; Rinehart, D.E.; Carlson, C.D.; Soderquist, C.Z.; Fadeff, S.K.

    1999-04-02

    Laboratory tests were performed to examine the efficacy of various leach treatments for decontaminating dissolver residual solids (KEACRESID1) produced during a 24-hour dissolution of K East Basin floor and Weasel Pit sludge composite in boiling 6 M HNO{sub 3}. The scope of this testing has been described in Section 4.5 of ''Testing Strategy to Support the Development of K Basin Sludge Treatment Process'' (Flament 1998). Radionuclides sorbed or associated with the residual solids generated in the K Basin sludge treatment process can restrict disposal of this solid to the Environmental Restoration Disposal Facility (ERDF). The starting dissolver residual solid for this testing, KEACRESID1, is a visibly heterogeneous material. This material contains radionuclides at concentrations above the ERDF Waste Acceptance Criteria for transuranics (TRU) by about a factor of 3, for {sup 239}Pu by a factor of 10, and for {sup 241}Am by a factor of 1.6. It meets the ERDF criterion for {sup 137}Cs by a factor of 4 and for uranium by a factor of 10. Therefore, the radionuclides of greatest interest in this leaching study are first {sup 239}Pu, and then {sup 241}Am, {sup 137}Cs, and uranium.

  20. An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

    PubMed Central

    Tavares, Eveline Queiroz de Pinho; Rubini, Marciano Regis; Mello-de-Sousa, Thiago Machado; Duarte, Gilvan Caetano; de Faria, Fabrícia Paula; Ferreira Filho, Edivaldo Ximenes; Kyaw, Cynthia Maria; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio Jose

    2013-01-01

    Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as Km = 27.5 ± 4.33 mg/mL, Vmax = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol. PMID:23936633

  1. Metabolic programming mediated by an essential fatty acid alters body composition and survival skills of a marine fish.

    PubMed

    Fuiman, Lee A; Perez, Kestrel O

    2015-11-22

    Metabolic programming occurs when variations in nutrition during a specific developmental window result in long-term metabolic effects. It has been studied almost exclusively in humans and other mammals but never in an ecological context. Here, we report metabolic programming and its functional consequences in a marine fish, red drum. We demonstrate that maternal provisioning of eggs with an essential fatty acid, docosahexaenoic acid (DHA), varies with DHA content of the maternal diet. When offspring are reared on a DHA-replete diet, whole-body DHA content of offspring depends upon the amount of DHA that was in the egg. We further demonstrate that whole-body DHA content is correlated with traits related to offspring fitness (escape responses, routine swimming, growth, and survival). DHA content of red drum eggs produced in nature is in the range where the effects of metabolic programming are most pronounced. Our findings indicate that during a brief developmental window, DHA plays a role in establishing the metabolic capacity for its own uptake or storage, with protracted and possibly permanent effects on ecologically important survival skills of individuals and important implications for dynamics of populations and food webs. PMID:26582018

  2. Effects of Essential Oils and Polyunsaturated Fatty Acids on Canine Skin Equivalents: Skin Lipid Assessment and Morphological Evaluation

    PubMed Central

    Cerrato, S.; Ramió-Lluch, L.; Fondevila, D.; Rodes, D.; Brazis, P.; Puigdemont, A.

    2013-01-01

    A canine skin equivalent model has been validated for the assessment of a topical formulation effects. Skin equivalents were developed from freshly isolated cutaneous canine fibroblasts and keratinocytes, after enzymatic digestion of skin samples (n = 8) from different breeds. Fibroblasts were embedded into a collagen type I matrix, and keratinocytes were seeded onto its surface at air-liquid interface. Skin equivalents were supplemented with essential oils and polyunsaturated fatty acid formulation or with vehicle. Skin equivalents were histopathologically and ultrastructurally studied, and the three main lipid groups (free fatty acids, cholesterol, and ceramides) were analyzed. Results showed that the culture method developed resulted in significant improvements in cell retrieval and confluence. Treated samples presented a thicker epidermis with increased number of viable cell layers, a denser and compact stratum corneum, and a more continuous basal membrane. Regarding lipid profile, treated skin equivalents showed a significant increase in ceramide content (51.7 ± 1.3) when compared to untreated (41.6  ±  1.4) samples. Ultrastructural study evidenced a compact and well-organized stratum corneum in both treated and control skin equivalents. In conclusion, cell viability and ceramides increase, after lipid supplementation, are especially relevant for the treatment of skin barrier disruptions occurring in canine atopic dermatitis. PMID:26464904

  3. Antilisterial effects of antibacterial formulations containing essential oils, nisin, nitrite and organic acid salts in a sausage model.

    PubMed

    Ghabraie, Mina; Vu, Khanh Dang; Huq, Tanzina; Khan, Avik; Lacroix, Monique

    2016-06-01

    This study was conducted to evaluate the effects of sixteen antibacterial formulations against Listeria monocytogenes in a sausage model using a standard experimental design with 4 independent factors at 2 levels (2(4)). Four independent factors consisted of nisin (12.5-25 ppm), nitrite (100-200 ppm) and organic acid salts (1.55-3.1 %) and the mixture of Chinese cinnamon and Cinnamon bark Essential Oils (EOs) (0.025-0.05 %). Based on the analysis, utilization of low (0.025 %) or high concentration (0.05 %) of EOs in combination with low concentration of nitrite (100 ppm), organic acid salts (1.55 %), and nisin (12.5 ppm) could reduce respectively 1.5 or 2.6 log CFU/g of L. monocytogenes in sausage at day 7 of storage as compared to the control. A predictive equation was created to predict the growth of L. monocytogenes in sausage. The sensory evaluation was then performed on selected optimized formulations in cooked meat (both pork and beef sausages) with a trained jury consisting of 35 individuals, demonstrated the selected antimicrobial formulations were organoleptically acceptable. The results revealed an important role of hurdle technology to control L. monocytogenes in meat product. PMID:27478218

  4. Synthesis and application of boronic acid-functionalized magnetic adsorbent for sensitive analysis of salbutamol residues in pig tissues.

    PubMed

    Fan, Hua; Wang, Chaozhan; Wei, Yinmao

    2015-12-01

    Salbutamol (SAL) is the most widely used β2 -agonist drug for asthma and chronic obstructive pulmonary patients, but it is also often abused as feed additive. In recent years, the abuse of SAL has led to a large number of food safety incidents. Therefore, the monitoring of SAL residues in animal products is very important. A highly selective boronate affinity magnetic adsorbent was synthesized and developed for detection of trace levels of SAL residues in pig tissue samples. The obtained Fe3O4@SiO2@FPBA(4-formylphenylboronic acid) magnetic adsorbent showed good adsorption ability to catechol and SAL, and then it was successfully applied as special magnetic solid-phase phase extraction adsorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous isolation and determination of cis-diol compounds. The binding capacity of catechol and SAL reached 96 and 50 µmol/g, respectively. The method was successfully established for the detection of trace levels of SAL in pig tissue samples. The linear range extended from 0.32 to 800 µg/kg (R(2) = 0.9994). The limit of detection of SAL was 0.19 µg/kg. The recoveries were satisfactory (89.5-108.0%) at three spiked levels with RSD between 2.1 and 11.3%. These results indicated that the method has potential for enrichment and detection of trace levels of SAL residual in animal food products. PMID:26061980

  5. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

    PubMed

    Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

    2016-03-01

    The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge. PMID:26722812

  6. The dominant detritus-feeding invertebrate in Arctic peat soils derives its essential amino acids from gut symbionts.

    PubMed

    Larsen, Thomas; Ventura, Marc; Maraldo, Kristine; Triadó-Margarit, Xavier; Casamayor, Emilio O; Wang, Yiming V; Andersen, Nils; O'Brien, Diane M

    2016-09-01

    Supplementation of nutrients by symbionts enables consumers to thrive on resources that might otherwise be insufficient to meet nutritional demands. Such nutritional subsidies by intracellular symbionts have been well studied; however, supplementation of de novo synthesized nutrients to hosts by extracellular gut symbionts is poorly documented, especially for generalists with relatively undifferentiated intestinal tracts. Although gut symbionts facilitate degradation of resources that would otherwise remain inaccessible to the host, such digestive actions alone cannot make up for dietary insufficiencies of macronutrients such as essential amino acids (EAA). Documenting whether gut symbionts also function as partners for symbiotic EAA supplementation is important because the question of how some detritivores are able to subsist on nutritionally insufficient diets has remained unresolved. To answer this poorly understood nutritional aspect of symbiont-host interactions, we studied the enchytraeid worm, a bulk soil feeder that thrives in Arctic peatlands. In a combined field and laboratory study, we employed stable isotope fingerprinting of amino acids to identify the biosynthetic origins of amino acids to bacteria, fungi and plants in enchytraeids. Enchytraeids collected from Arctic peatlands derived more than 80% of their EAA from bacteria. In a controlled feeding study with the enchytraeid Enchytraeus crypticus, EAA derived almost exclusively from gut bacteria when the worms fed on higher fibre diets, whereas most of the enchytraeids' EAA derived from dietary sources when fed on lower fibre diets. Our gene sequencing results of gut microbiota showed that the worms harbour several taxa in their gut lumen absent from their diets and substrates. Almost all gut taxa are candidates for EAA supplementation because almost all belong to clades capable of biosynthesizing EAA. Our study provides the first evidence of extensive symbiotic supplementation of EAA by microbial

  7. Nucleic acid labeling with ( sup 3 H)orotic acid and nucleotide profile in rats in protein deprivation, enteral and parenteral essential amino acid administration, and 5-fluorouracil treatment

    SciTech Connect

    Jakobsson, B.; el Hag, I.A.; Andersson, M.; Christensson, P.I.; Stenram, U. )

    1990-09-01

    Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of (3H)orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes.

  8. Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods. PMID:26381020

  9. Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

    PubMed

    Shiu, Jia-Hau; Chen, Chiu-Yueh; Chang, Long-Sen; Chen, Yi-Chun; Chen, Yen-Chin; Lo, Yu-Hui; Liu, Yu-Chen; Chuang, Woei-Jer

    2004-12-01

    Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins. PMID:15390258

  10. Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.

    PubMed

    Michel, K; Atkinson, P W

    2003-07-01

    For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family. PMID:12858343

  11. Enrichment of anhydrous milk fat in polyunsaturated fatty acid residues from linseed and rapeseed oils through enzymatic interesterification.

    PubMed

    Aguedo, Mario; Hanon, Emilien; Danthine, Sabine; Paquot, Michel; Lognay, Georges; Thomas, Annick; Vandenbol, Micheline; Thonart, Philippe; Wathelet, Jean-Paul; Blecker, Christophe

    2008-03-12

    Lipozyme TL IM was used in a solvent-free batch and microaqueous system for enzymatic interesterification of anhydrous milkfat (AMF) with linseed oil (LO) in binary blends and with rapeseed oil (RO) in one ternary blend. The aim was to obtain and characterize physicochemically fats enriched with unsaturated C 18 fatty acids (oleic, linoleic, and, especially, linolenic acids) from natural vegetable oils. Binary blends of AMF/LO 100/0, 90/10, 80/20, 70/30, and 60/40 (w/w) were interesterified. The change in triacylglycerol (TAG) profiles showed that quasi-equilibrium conditions were reached after 4-6 h of reaction. Free fatty acid contents <1%. The decrease in solid fat content and in dropping point temperature obtained with increasing content of LO and interesterification resulted in good plastic properties for the products originating from the blends 70/30 and 60/40. This was confirmed by textural measurements. Melting profiles determined by differential scanning calorimetry showed complete disappearance of low-melting TAGs from LO and the formation of intermediary species with a lower melting temperature. Oxidative stability of the interesterified products was diminished with increasing LO content, resulting in low oxidation induction times. A ternary blend composed of AMF/RO/LO 70/20/10 gave satisfactory rheological and oxidative properties, fulfilling the requirements for a marketable spread and, moreover, offering increased potential health benefits due to the enriched content in polyunsaturated fatty acid residues. PMID:18271538

  12. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  13. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    PubMed

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity. PMID:24880119

  14. Distribution of /sup 14/C after oral administration of (1-/sup 14/C)linoleic acid in rats fed different levels of essential fatty acids

    SciTech Connect

    Becker, W.

    1984-09-01

    Rats from an inbred Sprague-Dawley strain were fed semisynthetic diets with a low (0.3 energy percent (en %)), normal (3 en %) or high (10 en %) content of essential fatty acids (EFA) for at least three generations. Twenty-nine- to 33-day-old male rats were given a single intragastric dose of (1-14C)linoleic acid in olive oil, and the respiratory CO2, urine and feces were collected for 46 hours (expt 1) or 20 hours (expt 2). The 14C activity in respiratory CO2, feces, urine and the carcass was determined in both experiments. In experiment 2 it was also measured in samples of the brown fat, liver, adrenals, white fat, skeletal muscles and brain. In both experiments the rats fed the low EFA diet retained significantly more 14C activity than the rats fed the normal or high EFA diets. In all groups the concentration of label was highest in the brown fat and the adrenals, but the above differences among the groups with respect to 14C retention were mainly observed in the liver, skeletal muscles and brain.

  15. Nonenzymatic oligomerization reactions on templates containing inosinic acid or diaminopurine nucleotide residues

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1999-01-01

    The template-directed oligomerization of nucleoside-5'-phosphoro-2-methyl imidazolides on standard oligonucleotide templates has been studied extensively. Here, we describe experiments with templates in which inosinic acid (I) is substituted for guanylic acid, or 2,6-diaminopurine nucleotide (D) for adenylic acid. We find that the substitution of I for G in a template is strongly inhibitory and prevents any incorporation of C into internal positions in the oligomeric products of the reaction. The substitution of D for A, on the contrary, leads to increased incorporation of U into the products. We found no evidence for the template-directed facilitation of oligomerization of A or I through A-I base pairing. The significance of these results for prebiotic chemistry is discussed.

  16. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

  17. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

    PubMed Central

    Anderson, C W; Schmitt, R C; Smart, J E; Lewis, J B

    1984-01-01

    Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins. Images PMID:6323739

  18. [Correlation between the output and composition of essential oil and the level of salicylic acid in mint plants at different ontogenetic stages].

    PubMed

    Shelepova, O V; Kondrat'eva, V V; Voronkova, T V; Olekhnovich, L S

    2013-01-01

    Dynamic changes in the content of acetylsalicylic acid and the output and qualitative composition of essential oil have been studied in mint plants (Mentha spicata L. and cultivar Medichka) during their ontogenesis with allowance for changes in weather conditions. Ontogenetic changes in the level of acetylsalicylic acid in leaf tissues are found to be similar in both cv. Medichka and M. spicata. In the case of cv. Medichka, this change is connected with the dynamics of the production and the qualitative composition of essential oil; in the case of M. spicata, this connection is less expressed. The role of acetylsalicylic acid and essential oil in plant adaptation to the environment is discussed. PMID:24171311

  19. Chemical fragmentation by o-iodosobenzoic acid of. cap alpha. -chain of histidine decarboxylase from Micrococcus sp. n. at tryptophan residues

    SciTech Connect

    Alekseeva, E.A.; Grebenshchikova, O.G.; Prozorovskii, V.N.

    1987-02-10

    The carboxymethylated ..cap alpha..-chain of histidine decarboxylase from Micrococcus sp. n., which contains four tryptophan residues, was cleaved by o-iodosobenzoic acid. Five fragments were isolated in homogeneous form by means of gel filtration on Sephadex, rechromatography, and high-voltage paper electrophoresis. The molecular weight, amino acid composition, and N-terminal amino acid sequence were determined for all the peptides isolated.

  20. Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise.

    PubMed

    Moberg, Marcus; Apró, William; Ekblom, Björn; van Hall, Gerrit; Holmberg, Hans-Christer; Blomstrand, Eva

    2016-06-01

    Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo

  1. Dietary self-selection of protein-unbalanced diets supplemented with three essential amino acids in Nile tilapia.

    PubMed

    Fortes-Silva, R; Rosa, P V; Zamora, S; Sánchez-Vázquez, F J

    2012-02-01

    Animals do not eat whatever food item they encounter, but choose different foods that best match their requirements. Fish exhibit such "nutritional wisdom" and adapt their feeding behaviour and food intake according to their needs and the nutritional properties of diets. In this paper, we tested the ability of Nile tilapia to select between diets with a balanced or unbalanced composition of essential amino acids. To this end, three different diets were prepared: a gelatine based diet (D(1)), a gelatine diet supplemented with three essential amino acids (EAA, l-tryptophane, l-methionine, l-threonine) (D(2)), and a diet containing only cellulose and the three crystalline EAA (D(3)). In addition, the putative role of both orosensorial factors (using pellets vs capsules) and social interactions (single vs groups of ten fish) was investigated. To this end, a total of 68 male tilapia of about 141±48 g (mean±S.D.) were challenged, individually or in groups, to select between D(1)vs D(2) using pellets dispensed by self-feeders (exp. 1). In another experiment (exp. 2), 11 individual fish were challenged to select encapsulated diets with non flavour or smell proprieties (D(1)vs D(2)), and in exp. 3 fish were challenged to self-supplementation in EAA (D(1)vs D(3)). The results showed the ability of tilapia to avoid the EAA-deficient diet, choosing 82.2% D(2) in the case of individual fish, and 80.8% D(2) in the case of fish groups. Dietary selection was not directly driven by the orosensorial characteristics of food, since tilapia sustained a higher preference for D(2) when fed with encapsulated diets. Finally, in exp. 3 tilapia self-supplemented the EAA deficiency by selecting a synchronised combination of D(1) and D(3) that matched their nutritional requirements. These findings highlighted the capacity of fish to make dietary selection based on the EAA content, which should be considered when discussing food intake regulation mechanisms, and diet formulation and

  2. A role for residue 151 of LamB in bacteriophage lambda adsorption: possible steric effect of amino acid substitutions.

    PubMed

    Charbit, A; Werts, C; Michel, V; Klebba, P E; Quillardet, P; Hofnung, M

    1994-06-01

    LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda have been previously grouped in two classes. Class I mutants block growth of lambda with wild-type host range (lambda h+) but support growth of one-step extended-host-range mutants (lambda h). Class II mutants block lambda h but support growth of two-step extended host range mutants (lambda hh*). While Class I mutations occur at 11 different amino acid sites, in five distinct portions of LamB, all the Class II mutations analyzed previously correspond to the same G-to-D change at amino acid 151. We generated by in vitro mutagenesis four different new substitutions at site 151 (to S, V, R, and C). Two of the mutants (G-151-->V [G151V] and G151R) were of Class II, while the two others (G151S and G151C) were of Class I, demonstrating that not only the site but also the nature of the substitutions at residue 151 was critical for the phage sensitivity phenotypes. The introduction of a negatively charged, a positively charged, or an aliphatic nonpolar residue at site 151 of LamB prevented both lambda h+ and lambda h adsorption, indicating that the block is not due to a charge effect. In contrast to G151D, which was sensitive to all the lambda hh* phages, G151V and G151R conferred sensitivity to only four of the five lambda hh* phages. Thus, G151V and G151R represent a new subclass of Class II LamB mutations that is more restrictive with respect to the growth of lambda hh*. Our results agree with the hypothesis that residue 151 belongs to an accessibility gate controlling the access to the phage tight-binding site and that substitutions at this residue affect the access of the phage to the binding site in relation to the size of the substitute side chain (surface area): the most restrictive changes are G151V and G151R, followed to a lesser extent by G151D and they by G151S and G151C. PMID:8195074

  3. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

    PubMed Central

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-01-01

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane. PMID:27120610

  4. Influence of washing time on residual contamination of carcasses sprayed with lauric acid-potassium hydroxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of experiments were conducted to examine reductions in bacterial contamination of broiler carcasses washed for various times in a spray cabinet with a 2% lauric acid (LA)-1% potassium hydroxide (KOH) solution (w/v). Forty eviscerated carcasses and 5 ceca were obtained from the processing li...

  5. Influence of washing time on residual contamination of carcasses sprayed with lauric acid-potassium hydroxide.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of experiments were conducted to examine reductions in bacterial contamination of broiler carcasses washed for various times in a spray cabinet with a 2% lauric acid (LA)-1% potassium hydroxide (KOH) (w/v) solution. Forty eviscerated carcasses and 5 ceca were obtained from the processing l...

  6. Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

    SciTech Connect

    Pokkuluri, P. R.; Cai, X.; Raffen, R.; Gu, M.; Stevens, F. J.; Schiffer, M.

    2002-07-01

    Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

  7. Effect of the replacement of aspartic acid/glutamic acid residues with asparagine/glutamine residues in RNase He1 from Hericium erinaceus on inhibition of human leukemia cell line proliferation.

    PubMed

    Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Suzuki, Mamoru; Inokuchi, Norio

    2015-01-01

    RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity. PMID:25338779

  8. Effects of Feeding Garlic and Juniper Berry Essential Oils on Milk Fatty Acid Composition of Dairy Cows

    PubMed Central

    Yang, Wen Zhu; He, Mao Long

    2016-01-01

    Essential oils (EOs) from plant extracts have been reported to have an antibacterial activity against gram-negative and gram-positive bacteria. Several of the gram-positive bacteria are involved in ruminal biohydrogenation of fatty acids (FAs), thus suggesting that feeding EOs could lower biohydrogenation of FA because of a decrease in the number of bacteria involved in that process. As a result, milk FA profiles are expected to be modified. In addition, monensin was approved as an antibiotic to be fed in dairy cattle, and it was reported that dairy cows supplemented with monensin produced milk containing higher concentration of 18:1 t10 and 18:1 t11. The objective of this study was to evaluate the effects of two EOs (garlic and juniper berry oils) and monensin on FA profiles of milk fat. Four ruminally fistulated Holstein dairy cows were used in a 4 × 4 Latin square experiment. Cows were fed for ad libitum intake a total mixed ration without supplementation (control), or supplemented with monensin (330 mg/head per day), garlic oil (5 g/head per day), or juniper berry oil (2 g/head per day). The FA composition of saturated, monounsaturated, or polyunsaturated was not affected by supplementation of EO and monensin. However, proportion of conjugated linoleic acid trans 10, cis 12 (CLA t10, c12) was higher (P < 0.05) for cows fed EO or monensin than for control cows. Supplementation of monensin increased (P < 0.05) the proportion of total trans FA compared with the control. These results indicate that supplementation of the dairy cow diet with garlic or juniper berry EO or monensin had the potential to increase the proportion of CLA t10, c12 in milk fat with minimal overall effects on FA of milk fat. The results also confirm the increase of 18:1 t10 in milk fat by feeding monensin to dairy cows. PMID:27127411

  9. PGC-1 coactivators in β-cells regulate lipid metabolism and are essential for insulin secretion coupled to fatty acids

    PubMed Central

    Oropeza, Daniel; Jouvet, Nathalie; Bouyakdan, Khalil; Perron, Gabrielle; Ringuette, Lea-Jeanne; Philipson, Louis H.; Kiss, Robert S.; Poitout, Vincent; Alquier, Thierry; Estall, Jennifer L.

    2015-01-01

    Objectives Peroxisome proliferator-activated receptor γ coactivator 1 (PPARGCA1, PGC-1) transcriptional coactivators control gene programs important for nutrient metabolism. Islets of type 2 diabetic subjects have reduced PGC-1α expression and this is associated with decreased insulin secretion, yet little is known about why this occurs or what role it plays in the development of diabetes. Our goal was to delineate the role and importance of PGC-1 proteins to β-cell function and energy homeostasis. Methods We investigated how nutrient signals regulate coactivator expression in islets and the metabolic consequences of reduced PGC-1α and PGC-1β in primary and cultured β-cells. Mice with inducible β-cell specific double knockout of Pgc-1α/Pgc-1β (βPgc-1 KO) were created to determine the physiological impact of reduced Pgc1 expression on glucose homeostasis. Results Pgc-1α and Pgc-1β expression was increased in primary mouse and human islets by acute glucose and palmitate exposure. Surprisingly, PGC-1 proteins were dispensable for the maintenance of mitochondrial mass, gene expression, and oxygen consumption in response to glucose in adult β-cells. However, islets and mice with an inducible, β-cell-specific PGC-1 knockout had decreased insulin secretion due in large part to loss of the potentiating effect of fatty acids. Consistent with an essential role for PGC-1 in lipid metabolism, β-cells with reduced PGC-1s accumulated acyl-glycerols and PGC-1s controlled expression of key enzymes in lipolysis and the glycerolipid/free fatty acid cycle. Conclusions These data highlight the importance of PGC-1s in coupling β-cell lipid metabolism to promote efficient insulin secretion. PMID:26629405

  10. Identification of Ourmiavirus 30K movement protein amino acid residues involved in symptomatology, viral movement, subcellular localization and tubule formation.

    PubMed

    Margaria, Paolo; Anderson, Charles T; Turina, Massimo; Rosa, Cristina

    2016-09-01

    Several plant viruses encode movement proteins (MPs) classified in the 30K superfamily. Despite a great functional diversity, alignment analysis of MP sequences belonging to the 30K superfamily revealed the presence of a central core region, including amino acids potentially critical for MP structure and functionality. We performed alanine-scanning mutagenesis of the Ourmia melon virus (OuMV) MP, and studied the effects of amino acid substitutions on MP properties and virus infection. We identified five OuMV mutants that were impaired in systemic infection in Nicotiana benthamiana and Arabidopsis thaliana, and two mutants showing necrosis and pronounced mosaic symptoms, respectively, in N. benthamiana. Green fluorescent protein fusion constructs (GFP:MP) of movement-defective MP alleles failed to localize in distinct foci at the cell wall, whereas a GFP fusion with wild-type MP (GFP:MPwt) mainly co-localized with plasmodesmata and accumulated at the periphery of epidermal cells. The movement-defective mutants also failed to produce tubular protrusions in protoplasts isolated from infected leaves, suggesting a link between tubule formation and the ability of OuMV to move. In addition to providing data to support the importance of specific amino acids for OuMV MP functionality, we predict that these conserved residues might be critical for the correct folding and/or function of the MP of other viral species in the 30K superfamily. PMID:26637973

  11. Effect of jasmonic acid elicitation on the yield, chemical composition, and antioxidant and anti-inflammatory properties of essential oil of lettuce leaf basil (Ocimum basilicum L.).

    PubMed

    Złotek, Urszula; Michalak-Majewska, Monika; Szymanowska, Urszula

    2016-12-15

    The effect of elicitation with jasmonic acid (JA) on the plant yield, the production and composition of essential oils of lettuce leaf basil was evaluated. JA-elicitation slightly affected the yield of plants and significantly increased the amount of essential oils produced by basil - the highest oil yield (0.78±0.005mL/100gdw) was achieved in plants elicited with 100μM JA. The application of the tested elicitor also influenced the chemical composition of basil essential oils - 100μM JA increased the linalool, eugenol, and limonene levels, while 1μM JA caused the highest increase in the methyl eugenol content. Essential oils from JA-elicited basil (especially 1μM and 100μM) exhibited more effective antioxidant and anti-inflammatory potential; therefore, this inducer may be a very useful biochemical tool for improving production and composition of herbal essential oils. PMID:27451148

  12. Characterization of Active Packaging Films Made from Poly(Lactic Acid)/Poly(Trimethylene Carbonate) Incorporated with Oregano Essential Oil.

    PubMed

    Liu, Dong; Li, Hongli; Jiang, Lin; Chuan, Yongming; Yuan, Minglong; Chen, Haiyun

    2016-01-01

    Antimicromial and antioxidant bioactive films based on poly(lactic acid)/poly(trimenthylene carbonate) films incorporated with different concentrations of oregano essential oil (OEO) were prepared by solvent casting. The antimicrobial, antioxidant, physical, thermal, microstructural, and mechanical properties of the resulting films were examined. Scanning electron microscopy analysis revealed that the cross-section of films became rougher when OEO was incorporated into PLA/PTMC blends. Differential scanning calorimetry analysis indicated that crystallinity of PLA phase decreased by the addition of OEO, but this did not affect the thermal stability of the films. Water vapor permeability of films slightly increased with increasing concentration of OEO. However, active PLA/PTMC/OEO composite films showed adequate barrier properties for food packaging application. The antimicrobial and antioxidant capacities were significantly improved with the incorporation of OEO (p < 0.05). The results demonstrated that an optimal balance between the mechanical, barrier, thermal, antioxidant, and antimicrobial properties of the films was achieved by the incorporation of 9 wt % OEO into PLA/PTMC blends. PMID:27240336

  13. Essential Amino Acids and Exercise Tolerance in Elderly Muscle-Depleted Subjects with Chronic Diseases: A Rehabilitation without Rehabilitation?

    PubMed Central

    Aquilani, Roberto; D'Antona, Giuseppe; Baiardi, Paola; Iadarola, Paolo; Pasini, Evasio; Barbieri, Annalisa

    2014-01-01

    Exercise intolerance remains problematic in subjects with chronic heart failure (CHF) and/or chronic obstructive pulmonary disease (COPD). Recent studies show that supplemented essential amino acids (EAAs) may exert beneficial effects on CHF/COPD physical capacity. The results from 3 investigations (2 conducted on CHF and 1 on COPD subjects) served as the basis for this paper. The 3 studies consistently showed that elderly CHF and COPD improved exercise intolerance after 1–3 months of EAA supplementation (8 g/d). In CHF exercise capacity increased 18.7% to 23% (watts; bicycle test), and 12% to 22% (meters) in 6 min walking test. Moreover, patients reduced their resting plasma lactate levels (by 25%) and improved tissue insulin sensitivity by 16% (HOMA index). COPD subjects enjoyed similar benefits as CHF ones. They increased physical autonomy by 78.6% steps/day and decreased resting plasma lactate concentrations by 23%. EAA mechanisms explaining improved exercise intolerance could be increases in muscle aerobic metabolism, mass and function, and improvement of tissue insulin sensitivity (the latter only for the CHF population). These mechanisms could be accounted for by EAA's intrinsic physiological activity which increases myofibrils and mitochondria genesis in skeletal muscle and myocardium and glucose control. Supplemented EAAs can improve the physical autonomy of subjects with CHF/COPD. PMID:25009815

  14. Nicotinic Acid Adenine Dinucleotide Phosphate (Naadp+) Is an Essential Regulator of T-Lymphocyte Ca2+-Signaling

    PubMed Central

    Berg, Ingeborg; Potter, Barry V.L.; Mayr, Georg W.; Guse, Andreas H.

    2000-01-01

    Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP+) dose-dependently stimulated intracellular Ca2+-signaling. At a concentration of 10 nM NAADP+ evoked repetitive and long-lasting Ca2+-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca2+-peak followed by trains of smaller Ca2+-oscillations was observed. Higher concentrations of NAADP+ (1 and 10 μM) gradually reduced the initial Ca2+-peak, and a complete self-inactivation of Ca2+-signals was seen at 100 μM. The effect of NAADP+ was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4,5-trisphosphate– and cyclic adenosine diphosphoribose–mediated Ca2+-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP+. Most importantly, microinjection of a self-inactivating concentration of NAADP+ completely abolished subsequent stimulation of Ca2+-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP+ Ca2+-release system is essential for T-lymphocyte Ca2+-signaling. PMID:10931869

  15. Essential amino acids and exercise tolerance in elderly muscle-depleted subjects with chronic diseases: a rehabilitation without rehabilitation?

    PubMed

    Aquilani, Roberto; D'Antona, Giuseppe; Baiardi, Paola; Gambino, Arianna; Iadarola, Paolo; Viglio, Simona; Pasini, Evasio; Verri, Manuela; Barbieri, Annalisa; Boschi, Federica

    2014-01-01

    Exercise intolerance remains problematic in subjects with chronic heart failure (CHF) and/or chronic obstructive pulmonary disease (COPD). Recent studies show that supplemented essential amino acids (EAAs) may exert beneficial effects on CHF/COPD physical capacity. The results from 3 investigations (2 conducted on CHF and 1 on COPD subjects) served as the basis for this paper. The 3 studies consistently showed that elderly CHF and COPD improved exercise intolerance after 1-3 months of EAA supplementation (8 g/d). In CHF exercise capacity increased 18.7% to 23% (watts; bicycle test), and 12% to 22% (meters) in 6 min walking test. Moreover, patients reduced their resting plasma lactate levels (by 25%) and improved tissue insulin sensitivity by 16% (HOMA index). COPD subjects enjoyed similar benefits as CHF ones. They increased physical autonomy by 78.6% steps/day and decreased resting plasma lactate concentrations by 23%. EAA mechanisms explaining improved exercise intolerance could be increases in muscle aerobic metabolism, mass and function, and improvement of tissue insulin sensitivity (the latter only for the CHF population). These mechanisms could be accounted for by EAA's intrinsic physiological activity which increases myofibrils and mitochondria genesis in skeletal muscle and myocardium and glucose control. Supplemented EAAs can improve the physical autonomy of subjects with CHF/COPD. PMID:25009815

  16. Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Kjeldsen, Frank; Haselmann, Kim F; Sørensen, Esben S; Zubarev, Roman A

    2003-03-15

    In hot electron capture dissociation (HECD), multiply protonated polypeptides fragment upon capturing approximately 11-eV electrons. The excess of energy upon the primary c, z* cleavage induces secondary fragmentation in z* fragments. The resultant w ions allow one to distinguish between the isomeric Ile and Leu residues. The analytical utility of HECD is evaluated using tryptic peptides from the bovine milk protein PP3 containing totally 135 amino acid residues. Using a formal procedure for Ile/Leu (Xle) residue assignment, the identities of 20 out of 25 Xle residues (80%) were determined. The identity of an additional two residues could be correctly guessed from the absence of the alternative w ions, and only two residues, for which neither expected nor alternative w ions were observed, remained unassigned. Reinspection of conventional ECD spectra also revealed the presence of Xle w ions, although at lower abundances, with 44% of all Xle residues distinguished. Using a dispenser cathode as an electron source, identification of four out of five Xle residues in a 2.7-kDa peptide was possible with one acquisition 2 s long, with identification of all five residues by averaging of five such acquisitions. Unlike the case of high-energy collision-induced dissociation, no d ions were observed in the HECD of tryptic peptides. PMID:12659185

  17. The Last C-Terminal Residue of VP3, Glutamic Acid 257, Controls Capsid Assembly of Infectious Bursal Disease Virus

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Da Costa, Bruno; Delmas, Bernard

    2004-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH2-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process. PMID:15016850

  18. Prediction of Residue Status to Be Protected or Not Protected From Hy-drogen Exchange Using Amino Acid Sequence Only.

    PubMed

    Nikita V, Dovidchenko; Oxana V, Galzitskaya

    2008-01-01

    We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of being folded in the equilibrium intermediate or native state and will be protected from hydrogen exchange using amino acid sequence only. Our approach FoldUnfold for the prediction of unstructured regions has been applied to seven different proteins. For 80% of the residues considered in this paper we can predict correctly their status: will they be protected or not from hydrogen exchange. An additional goal of our study is to assess whether properties inferred using the bioinformatics approach are easily applicable to predict behavior of proteins in solution. PMID:18949078

  19. Prediction of Residue Status to Be Protected or Not Protected From Hy-drogen Exchange Using Amino Acid Sequence Only

    PubMed Central

    Dovidchenko, Nikita V; Galzitskaya, Oxana V

    2008-01-01

    We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of being folded in the equilibrium intermediate or native state and will be protected from hydrogen exchange using amino acid sequence only. Our approach FoldUnfold for the prediction of unstructured regions has been applied to seven different proteins. For 80% of the residues considered in this paper we can predict correctly their status: will they be protected or not from hydrogen exchange. An additional goal of our study is to assess whether properties inferred using the bioinformatics approach are easily applicable to predict behavior of proteins in solution. PMID:18949078

  20. [Determination of glyphosate and aminomethylphosphonic acid residues in foods using high performance liquid chromatography-mass spectrometry/mass spectrometry].

    PubMed

    Li, Bo; Deng, Xiaojun; Guo, Dehua; Jin, Shuping

    2007-07-01

    A method for the determination of glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in plant products, such as rice, wheat, vegetables, fruits and tea, pig and chicken muscles, aquatic products, chestnut, honey, etc., was developed using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). In this method, PMG and AMPA were extracted with water from samples, defatted using an extraction step with dichloromethane, and purified using a cation-exchange (CAX) solid phase extraction cartridge. Then, these were derived using fluorenylmethylchloroformate (FMOC-Cl) in borate buffer for subsequent HPLC-MS/MS analysis. Isotope-labeled PMG 1, 2(13)- C(15) N was used as the internal standard for the quantitative analysis of two residues. For all samples, the recoveries ranged from 80.0% to 104% and the relative standard deviations (RSDs) ranged from 6.7% to 18.2%. The limit of quantification (LOQ) was determined to be 0.05 mg/kg with a linear range of 0.20-10 microg/L. It is demonstrated that this method is reliable and sensitive for the analysis of PMG and APMA with low concentrations in foods. PMID:17970103

  1. Lead Isotope Compositions of Acid Residues from Olivine-Phyric Shergottite Tissint: Implications for Heterogeneous Shergottite Source Reservoirs

    NASA Technical Reports Server (NTRS)

    Moriwaki, R.; Usui, T.; Yokoyama, T.; Simon, J. I.; Jones, J. H.

    2015-01-01

    Geochemical studies of shergottites suggest that their parental magmas reflect mixtures between at least two distinct geochemical source reservoirs, producing correlations between radiogenic isotope compositions and trace element abundances. These correlations have been interpreted as indicating the presence of a reduced, incompatible element- depleted reservoir and an oxidized, incompatible- element-enriched reservoir. The former is clearly a depleted mantle source, but there is ongoing debate regarding the origin of the enriched reservoir. Two contrasting models have been proposed regarding the location and mixing process of the two geochemical source reservoirs: (1) assimilation of oxidized crust by mantle derived, reduced magmas, or (2) mixing of two distinct mantle reservoirs during melting. The former requires the ancient Martian crust to be the enriched source (crustal assimilation), whereas the latter requires isolation of a long-lived enriched mantle domain that probably originated from residual melts formed during solidification of a magma ocean (heterogeneous mantle model). This study conducts Pb isotope and trace element concentration analyses of sequential acid-leaching fractions (leachates and the final residues) from the geochemically depleted olivine-phyric shergottite Tissint. The results suggest that the Tissint magma is not isotopically uniform and sampled at least two geochemical source reservoirs, implying that either crustal assimilation or magma mixing would have played a role in the Tissint petrogenesis.

  2. Molecular mechanism of long-range synergetic color tuning between multiple amino acid residues in conger rhodopsin

    PubMed Central

    Watanabe, Hiroshi C.; Mori, Yoshiharu; Tada, Takashi; Yokoyama, Shozo; Yamato, Takahisa

    2011-01-01

    The synergetic effects of multiple rhodopsin mutations on color tuning need to be completely elucidated. Systematic genetic studies and spectroscopy have demonstrated an interesting example of synergetic color tuning between two amino acid residues in conger rhodopsin's ancestral pigment (p501): —a double mutation at one nearby and one distant residue led to a significant λmax blue shift of 13 nm, whereas neither of the single mutations at these two sites led to meaningful shifts. To analyze the molecular mechanisms of this synergetic color tuning, we performed homology modeling, molecular simulations, and electronic state calculations. For the double mutant, N195A/A292S, in silico mutation analysis demonstrated conspicuous structural changes in the retinal chromophore, whereas that of the single mutant, A292S, was almost unchanged. Using statistical ensembles of QM/MM optimized structures, the excitation energy of retinal chromophore was evaluated for the three visual pigments. As a result, the λmax shift of double mutant (DM) from p501 was –8 nm, while that of single mutant (SM) from p501 was +1 nm. Molecular dynamics simulation for DM demonstrated frequent isomerization between 6-s-cis and 6-s-trans conformers. Unexpectedly, however, the two conformers exhibited almost identical excitation energy, whereas principal component analysis (PCA) identified the retinal-counterion cooperative change of BLA (bond length alternation) and retinal-counterion interaction lead to the shift. PMID:21297892

  3. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets

    PubMed Central

    Palermo, Giulia; Bauer, Inga; Campomanes, Pablo; Cavalli, Andrea; Armirotti, Andrea; Girotto, Stefania; Rothlisberger, Ursula; De Vivo, Marco

    2015-01-01

    The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts. PMID:26111155

  4. The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease

    PubMed Central

    Lugo-Martinez, Jose; Pejaver, Vikas; Pagel, Kymberleigh A.; Mort, Matthew; Cooper, David N.; Mooney, Sean D.; Radivojac, Predrag

    2016-01-01

    Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption. PMID:27564311

  5. Diminishing of aggregation for bovine liver catalase through acidic residues modification.

    PubMed

    Hashemnia, S; Moosavi-Movahedi, A A; Ghourchian, H; Ahmad, F; Hakimelahi, G H; Saboury, A A

    2006-12-15

    The tendency of proteins to aggregate is an important problem in biotechnology and the pharmaceutical industry. Because proteins in the aggregated state generally do not have the same biological activity as proteins in the native state. In order to prevent aggregation, it is essential to know the effective parameters in anti-aggregation mechanism. Using a chemical protein modification approach, UV-vis and fluorescence spectroscopies and circular dichroism spectropolarimetry, this study investigates the parameters involved in anti-aggregation mechanism of bovine liver catalase. Our findings clearly indicate that the modified bovine liver catalase provides better protection than the native enzyme against thermal aggregation. It seems that a decrease in hydrophobicity resulting in chemical modification plays an important role in preventing aggregation. PMID:16828155

  6. Just three water molecules can trigger the undesired nonenzymatic reactions of aspartic acid residues: new insight from a quantum-chemical study

    NASA Astrophysics Data System (ADS)

    Takahashi, O.

    2014-03-01

    Aspartic acid (Asp) residues in peptides and proteins (L-Asp) can undergo spontaneous, nonenzymatic reactions under physiological conditions by which abnormal L-β-Asp, D-Asp, and/or D-β-Asp residues are formed. These altered Asp residues may affect the three-dimensional structures of the peptides and proteins and hence their properties and functions. In fact, the altered Asp residues are relevant to age-related diseases such as cataract and Alzheimer's disease. Most of the above reactions of the L-Asp residue proceed via a cyclic succinimide intermediate. In this paper, I propose a detailed mechanism of cyclization of an Asp residue (forming a precursor of the succinimide) by the B3LYP/6-31+G(d,p) density functional theory calculations carried out for a small Asp-containing model compound complexed with three water molecules which act as general acid-base catalysts in proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form. Then, successive reorientation of a water molecule and conformational change occur followed by the nucleophilic attack of the iminol nitrogen atom on the carboxyl carbon atom of the Asp side chain to form a five-membered ring. A satisfactory agreement was obtained between the calculated and experimental energetics.

  7. Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides

    PubMed Central

    Du, Yuzhe; Lee, Jung-Eun; Nomura, Yoshiko; Zhang, Tianxiang; Zhorov, Boris S.; Dong, Ke

    2011-01-01

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNav sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNav channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the α-cyano group at the phenoxybenzyl alcohol) and three type II (containing the α-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4–IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4–IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix. PMID:19154185

  8. Trace metal occurrences in acid-insoluble residues of the Ordovician Galena Group, southeastern Minnesota

    SciTech Connect

    Lively, R.S.; Mossler, J.H.; Morey, G.B. ); Hauck, S.A. . Natural Resources Research Inst.)

    1993-03-01

    Regional geochemical, studies on insoluble residues from Paleozoic carbonate rocks have become an integral part of the search for new Upper Mississippi Valley-type mineral deposits in the northern Midcontinent. The authors have extended these studies to southeastern Minnesota, an area well to the north of known lead-zinc deposits of commercial size and grade. In this region, a thin sequence of Upper Cambrian to Middle Ordovician strata unconformably overlies a complex Precambrian basement. More than 500 samples of limestone and dolomite from 40 drill holes and outcrops were analyzed for 29 related trace elements. Preliminary interpretations are based on the analysis of 380 samples of the Ordovician Galena Group from 37 localities. Results indicate that anomalous concentrations of Pb, Cu, zn, As, Cd, and Ni are confined to the southern half of the Galena subgroup area and extend less than 30 miles north of the Iowa border. The anomalous areas, as well as saddles between the, have a distinct northwest trend, coincident with structural features previously recognized in the Precambrian basement. The spatial relationships of the anomalies and the lack of direct correlation imply deposition from fluids moving north out of the main lead-zinc district along structural pathways. The lack of significant anomalies in the northern part of the subcrop area implies northwest weakening of the forces driving the metal-bearing fluids, as well as a decrease over distance in the absolute metal content of the migrating fluids.

  9. His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

    PubMed

    Koszelak-Rosenblum, Mary; Krol, Adam C; Simmons, Danielle M; Goulah, Christopher C; Wroblewski, Liliana; Malkowski, Michael G

    2008-09-01

    Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis. PMID:18596034

  10. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  11. Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids.

    PubMed

    Ma, Ying; Thota, Bala N S; Haag, Rainer; Budisa, Nediljko

    2015-11-15

    Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein-dendrimer conjugates with the defined architectures. We incorporated Azidohomoalanine (Aha) as methionine substitute in vivo into various protein scaffolds to allow non-invasive dendrimer conjugations (dendronylation). Our approach makes recombinant proteins accessible for the design of multivalent dendrimer conjugates since it enables the preparation of many sequences with various positions for regioselective dendronylation. PMID:26483199

  12. ACID EVAPORATION OF ULTIMA GOLD TM AB LIQUID SCINTILLATION COCKTAIL RESIDUE

    SciTech Connect

    Kyser, E.; Fondeur, F.; Crump, S.

    2011-12-21

    Prior analyses of samples from the F/H Lab solutions showed the presence of diisopropylnapthalene (DIN), a major component of Ultima Gold{trademark} AB liquid scintillation cocktail (LSC). These solutions are processed through H-Canyon Tank 10.5 and ultimately through the 17.8E evaporator. Similar solutions originated in SRNL streams sent to the same H Canyon tanks. This study examined whether the presence of these organics poses a process-significant hazard for the evaporator. Evaporation and calorimetry testing of surrogate samples containing 2000 ppm of Ultima Gold{trademark} AB LSC in 8 M nitric acid have been completed. These experiments showed that although reactions between nitric acid and the organic components do occur, they do not appear to pose a significant hazard for runaway reactions or generation of energetic compounds in canyon evaporators. The amount of off-gas generated was relatively modest and appeared to be well within the venting capacity of the H-Canyon evaporators. A significant fraction of the organic components likely survives the evaporation process primarily as non-volatile components that are not expected to represent any new process concerns during downstream operations such as neutralization. Laboratory Waste solutions containing minor amounts of DIN can be safely received, stored, transferred, and processed through the canyon waste evaporator.

  13. Dilute Sulfuric Acid Pretreatment of Agricultural and Agro-Industrial Residues for Ethanol Production

    NASA Astrophysics Data System (ADS)

    Martin, Carlos; Alriksson, Björn; Sjöde, Anders; Nilvebrant, Nils-Olof; Jönsson, Leif J.

    The potential of dilute-acid prehydrolysis as a pretreatment method for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks was investigated. The prehydrolysis was performed at 122°C during 20, 40, or 60 min using 2% H2SO4 at a solid-to-liquid ratio of 1∶10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the prehydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/L, respectively, and a xylan conversion of more than 80%. High-glucose concentrations (26-33.5 g/L) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on prehydrolysis, indicating that the conditions were not severe enough to hydrolyze the hemicelluloses in these materials quantitatively. All prehydrolysates were readily fermentable by Saccharomyces cerevisiae. The dilute-acid prehydrolysis resulted in a 2.7-to 3.7-fold increase of the enzymatic convertibility of bagasse, but was not efficient for improving the enzymatic hydrolysis of peanut shells, cassava stalks, or rice hulls.

  14. /sup 113/Cd NMR studies of a 1:1 Cd adduct with an 18-residue finger peptide from HIV-1 nucleic acid binding protein, p7

    SciTech Connect

    South, T.L.; Kim, B.; Summers, M.F.

    1989-01-04

    The Zn/sup 2+/ and Cd/sup 2+/ adducts with the 18-residue peptide comprising the amino acid sequence of the first finger (residues 13 through 30) of retroviral nucleic acid binding proteins p7 from HIV-1 (the causative agent of AIDS) have been prepared. /sup 1/H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The /sup 113/Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures.

  15. Modeling the survival of Salmonella on slice cooked ham as a function of apple skin polyphenols, acetic acid, oregano essential oil and carvacrol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Response surface methodology was applied to investigate the combined effect of apple skin polyphenols (ASP), acetic acid (AA), oregano essential oil (O) and carvacrol (C) on the inactivation of Salmonella on sliced cooked ham. A full factorial experimental design was employed with control variables ...

  16. Determining the Optimum Dietary Tryptophan to Lysine Ratio in Growing Pigs Fed Diets Formulated with Hhigher Levels of Other Essential Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies on amino acid (AA) ratios require the first limiting AA (generally Lys) to be set below the requirement estimate. Graded levels of the AA being investigated are then fed to determine the required ratio. Essential AA (EAA) not under investigation are often set at their presumed requirement ra...

  17. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.

    PubMed

    Herr, F M; Aronson, J; Storch, J

    1996-01-30

    The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K

  18. Role of the glutamic acid 54 residue in transthyretin stability and thyroxine binding.

    PubMed

    Miyata, Masanori; Sato, Takashi; Mizuguchi, Mineyuki; Nakamura, Teruya; Ikemizu, Shinji; Nabeshima, Yuko; Susuki, Seiko; Suwa, Yoshiaki; Morioka, Hiroshi; Ando, Yukio; Suico, Mary Ann; Shuto, Tsuyoshi; Koga, Tomoaki; Yamagata, Yuriko; Kai, Hirofumi

    2010-01-12

    Transthyretin (TTR) is a tetrameric protein associated with amyloidosis caused by tetramer dissociation and monomer misfolding. The structure of two TTR variants (E54G and E54K) with Glu54 point mutation that cause clinically aggressive amyloidosis remains unclear, although amyloidogenicity of artificial triple mutations (residues 53-55) in beta-strand D had been investigated. Here we first analyzed the crystal structures and biochemical and biophysical properties of E54G and E54K TTRs. The direction of the Lys15 side chain in E54K TTR and the surface electrostatic potential in the edge region in both variants were different from those of wild-type TTR. The presence of Lys54 leads to destabilization of tetramer structure due to enhanced electrostatic repulsion between Lys15 of two monomers. Consistent with structural data, the biochemical analyses demonstrated that E54G and E54K TTRs were more unstable than wild-type TTR. Furthermore, the entrance of the thyroxine (T(4)) binding pocket in TTR was markedly narrower in E54K TTR and wider in E54G TTR compared with wild-type TTR. The tetramer stabilization and amyloid fibril formation assays in the presence of T(4) showed lower tetramer stability and more fibril formation in E54K and E54G TTRs than in wild-type TTR, suggesting decreased T(4) binding to the TTR variants. These findings indicate that structural modification by Glu54 point mutation may sufficiently alter tetramer stability and T(4) binding. PMID:19950966

  19. K30, H150, and H168 are essential residues for coordinating pyridoxal 5'-phosphate of O-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans.

    PubMed

    Zheng, Chunli; Nie, Li; Qian, Lin; Wang, Zhilou; Liu, Guizhen; Liu, Jianshe

    2010-06-01

    O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5'-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization. PMID:20033172

  20. The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis.

    PubMed

    Prakash, A; Kasbekar, D P

    2002-01-01

    Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor. We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa. The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N. crassa mutant ( erg-3). The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae. The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH. PMID:11810252

  1. N-terminal basic amino acid residues of Beet black scorch virus capsid protein play a critical role in virion assembly and systemic movement

    PubMed Central

    2013-01-01

    Background Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly. Results In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement. Conclusions We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP. PMID:23786675

  2. Addition of glutamine to essential amino acids and carbohydrate does not enhance anabolism in young human males following exercise.

    PubMed

    Wilkinson, Sarah B; Kim, Paul L; Armstrong, David; Phillips, Stuart M

    2006-10-01

    We examined the effect of a post-exercise oral carbohydrate (CHO, 1 g.kg(-1).h(-1)) and essential amino acid (EAA, 9.25 g) solution containing glutamine (0.3 g/kg BW; GLN trial) versus an isoenergetic CHO-EAA solution without glutamine (control, CON trial) on muscle glycogen resynthesis and whole-body protein turnover following 90 min of cycling at 65% VO2 peak. Over the course of 3 h of recovery, muscle biopsies were taken to measure glycogen resynthesis and mixed muscle protein synthesis (MPS), by incorporation of [ring-2H5] phenylalanine. Infusion of [1-13C] leucine was used to measure whole-body protein turnover. Exercise resulted in a significant decrease in muscle glycogen (p < 0.05) with similar declines in each trial. Glycogen resynthesis following 3 h of recovery indicated no difference in total accumulation or rate of repletion. Leucine oxidation increased 2.5 fold (p < 0.05) during exercise, returned to resting levels immediately post-exercise,and was again elevated at 3 h post-exercise (p < 0.05). Leucine flux, an index of whole-body protein breakdown rate, was reduced during exercise, but increased to resting levels immediately post-exercise, and was further increased at 3 h post-exercise (p < 0.05), but only during the CON trial. Exercise resulted in a marked suppression of whole-body protein synthesis (50% of rest; p < 0.05), which was restored post-exercise; however, the addition of glutamine did not affect whole-body protein synthesis post-exercise. The rate of MPS was not different between trials. The addition of glutamine to a CHO + EAA beverage had no effect on post-exercise muscle glycogen resynthesis or muscle protein synthesis, but may suppress a rise in whole-body proteolysis during the later stages of recovery. PMID:17111006

  3. Nutrient balancing of the adult worker bumblebee (Bombus terrestris) depends on the dietary source of essential amino acids

    PubMed Central

    Stabler, Daniel; Paoli, Pier P.; Nicolson, Susan W.; Wright, Geraldine A.

    2015-01-01

    ABSTRACT Animals carefully regulate the amount of protein that they consume. The quantity of individual essential amino acids (EAAs) obtained from dietary protein depends on the protein source, but how the proportion of EAAs in the diet affects nutrient balancing has rarely been studied. Recent research using the Geometric Framework for Nutrition has revealed that forager honeybees who receive much of their dietary EAAs from floral nectar and not from solid protein have relatively low requirements for dietary EAAs. Here, we examined the nutritional requirements for protein and carbohydrates of foragers of the buff-tailed bumblebee Bombus terrestris. By using protein (sodium caseinate) or an equimolar mixture of the 10 EAAs, we found that the intake target (nutritional optimum) of adult workers depended on the source and proportion of dietary EAAs. When bees consumed caseinate-containing diets in a range of ratios between 1:250 and 1:25 (protein to carbohydrate), they achieved an intake target (IT) of 1:149 (w/w). In contrast to those fed protein, bees fed the EAA diets had an IT more biased towards carbohydrates (1:560 w/w) but also had a greater risk of death than those fed caseinate. We also tested how the dietary source of EAAs affected free AAs in bee haemolymph. Bees fed diets near their IT had similar haemolymph AA profiles, whereas bees fed diets high in caseinate had elevated levels of leucine, threonine, valine and alanine in the haemolymph. We found that like honeybees, bumblebee workers prioritize carbohydrate intake and have a relatively low requirement for protein. The dietary source of EAAs influenced both the ratio of protein/EAA to carbohydrate and the overall amount of carbohydrate eaten. Our data support the idea that EAAs and carbohydrates in haemolymph are important determinants of nutritional state in insects. PMID:25617453

  4. Can 13C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes?

    PubMed Central

    Jones, Susan C.; Sabree, Zakee L.

    2015-01-01

    Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ13CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following 13C stable isotope analysis. Termite carcass samples (−27.0 ± 0.4‰, mean ± s.e.) were significantly different from termite gut filtrate samples (−27.53 ± 0.5‰), but not the wood diet (−26.0 ± 0.5‰) (F(2,64) = 6, P < 0.0052). δ13CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ13CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function. PMID:26336647

  5. Variable clinical manifestations of a glycine to glutamic acid substitution of the COL3A1 gene at residue 736

    SciTech Connect

    Pope, F.M.; Narcisi, P.; Richards, A.J.

    1994-09-01

    Glycine substitutions at the 3{prime} end of the COL3A1 gene generally produce a characteristic clinical phenotype including acrogeria and severe vascular fragility. Here we report a three generation British family in which the propositus presented with aneurysms of the groins. He, his mother, sister and elder daughter all had the external clinical phenotype of vascular EDS IV whilst another daughter and nephew were clinically normal. Cultured skin fibroblasts from the propositus and his clinically affected relatives poorly secreted normal and overmodified collagen III species. Normal components of secreted proteins predominated whilst overmodified molecules were prominent in intracellular material. Surprisingly the normal children also secreted less collagen type III than expected (though more than their clinically abnormal relatives). cDNA from bases 2671 to 3714 were amplified as four overlapping PCR fragments and analysed by DGGE. The region between 2671 and 3015 was heterozygous. Sequencing showed a mutation of glycine to glutamic acid at residue 736. This mutation created an extra Apa 1 restriction site which was suitable for family studies. These showed inheritance of the mutant gene by both vascular and non-vascular clinical phenotypes. This family therefore illustrates that replacement of glycine to glutamic acid at position 736 produces variable clinical and biochemical phenotypes ranging from easily recognizable vascular EDS IV with very poor collagen secretion to an EDS III-like picture and with less severe protein disturbance. The reasons for these differences are at present unexplained.

  6. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

    PubMed

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  7. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

    PubMed Central

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  8. Leaching and selective copper recovery from acidic leachates of Três Marias zinc plant (MG, Brazil) metallurgical purification residues.

    PubMed

    Sethurajan, Manivannan; Huguenot, David; Lens, Piet N L; Horn, Heinrich A; Figueiredo, Luiz H A; van Hullebusch, Eric D

    2016-07-15

    Zinc plant purification residue (ZPR), a typical Zn-hydrometallurgical waste, was collected from the Três Marias Zn plant (MG, Brazil). ZPR was characterized for its metal content and fractionation, mineralogy, toxicity and leachability. Toxicity characteristics leaching procedure (TCLP) and European Community Bureau of Reference (BCR) sequential extraction results revealed that this ZPR displays high percentages of metals (Cd, Cu, Zn and Pb) in the highly mobilizable fractions, increasing its hazardous potential. Bulk chemical analysis, pH dependent leaching and acid (H2SO4) leaching studies confirm that the ZPR is polymetallic, rich in Cd, Cu and Zn. The sulfuric acid concentration (1 M), agitation speed (450 rpm), temperature (40 °C) and pulp density (20 g L(-1)) were optimized to leach the maximum amount of heavy metals (Cd, Cu and Zn). Under optimum conditions, more than 50%, 70% and 60% of the total Cd, Cu and Zn present in the ZPR can be leached, respectively. The metals in the acid leachates were investigated for metal sulfide precipitation with an emphasis on selective Cu recovery. Metal sulfide precipitation process parameters such as initial pH and Cu to sulfide ratio were optimized as pH 1.5 and 1:0.5 (Cu:sulfide) mass ratio, respectively. Under optimum conditions, more than 95% of Cu can be selectively recovered from the polymetallic ZPR leachates. The Cu precipitates characterization studies reveal that they are approximately 0.1 μm in diameter and mainly consist of Cu and S. XRD analysis showed covellite (CuS), chalcanthite (CuSO4·5H2O) and natrochalcite (NaCu2(SO4)2(OH)·H2O) as the mineral phases. ZPRs can thus be considered as an alternative resource for copper production. PMID:27074201

  9. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

    PubMed

    Dempsey, Daniel R; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J

    2015-11-01

    Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis. PMID:26476413

  10. The adsorption of chromium (VI) from industrial wastewater by acid and base-activated lignocellulosic residues.

    PubMed

    Alvarez, Patricia; Blanco, Clara; Granda, Marcos

    2007-06-01

    This study deals with the adsorption of Cr(VI) from synthetic and industrial wastewater, produced by a sewage plant. The activated carbons were prepared from a lignocellulosic raw material by thermal treatment at 450 and 650 degrees C in the presence of acid (AlCl(3), HCl, H(3)PO(4) and H(2)SO(4)) and base (NaOH) agents. To optimize the adsorption of Cr(VI), the chemical modifications caused by each activating agent (related to the capability of Cr(VI) removal), and the optimal experimental conditions of the pH, Cr(VI) concentration, adsorbent dose and residence time, were studied. Thus, treatment with H(3)PO(4) gives rise to carbons with a high surface area and high efficiency for Cr(VI) removal at short equilibrium times. In contrast, the generation of active surface sites by means of NaOH requires longer equilibrium times, the adsorption being less effective than in the former case. The adsorption isotherms obey the Langmuir equation only in the first stages of the reaction but fit the Freundlich equations over the whole range studied, so the heat of adsorption can be easily calculated. The results also show that the activated carbons obtained can be recovered by filtration with an efficiency of 30% in the third cycle. PMID:17126488

  11. Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons

    PubMed Central

    Saito, Kazuki; Ito, Koichi

    2015-01-01

    In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome. PMID:25897120

  12. Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: changes in residual sugars and water-soluble organic acids during ripening.

    PubMed

    Upreti, P; McKay, L L; Metzger, L E

    2006-02-01

    Cheddar cheese ripening involves the conversion of lactose to glucose and galactose or galactose-6-phosphate by starter and nonstarter lactic acid bacteria. Under ideal conditions (i.e., where bacteria grow under no stress of pH, water activity, and salt), these sugars are mainly converted to lactic acid. However, during ripening of cheese, survival and growth of bacteria occurs under the stressed condition of low pH, low water activity, and high salt content. This forces bacteria to use alternate biochemical pathways resulting in production of other organic acids. The objective of this study was to determine if the level and type of organic acids produced during ripening was influenced by calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), lactose at pressing (2.4 vs. 0.78%), and S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for organic acids (citric, orotic, pyruvic, lactic, formic, uric, acetic, propanoic, and butyric acids) and residual sugars (lactose, galactose) during 48 wk of ripening using an HPLC-based method. Different factors influenced changes in concentration of residual sugars and organic acids during ripening and are discussed in detail. Our results indicated that the largest decrease in lactose and the largest increase in lactic acid occurred between salting and d 1 of ripening. It was interesting to observe that although the lactose content in cheese was influenced by several factors (Ca and P, residual lactose, and S/M), the concentration of lactic acid was influenced only by S/M. More lactic acid was produced in low S/M treatments compared with high S/M treatments. Although surprising for Cheddar cheese, a substantial amount (0.2 to 0.4%) of galactose was observed throughout ripening in all treatments. Minor changes in the levels of citric, uric, butyric, and propanoic acids were observed during

  13. Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme.

    PubMed

    el-Maghrabi, M R; Pate, T M; D'Angelo, G; Correia, J J; Lively, M O; Pilkis, S J

    1987-08-25

    The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which

  14. Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase†

    PubMed Central

    Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N.; Perera, Lalith; Stafford, Darrel W.; Straight, David L.

    2009-01-01

    We used recombinant techniques to create a two-chain form (residues 1–345 and residues 346–758) of the vitamin K-dependent γ-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase’s transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain. PMID:18498174

  15. Hepatotoxicity of Pentavalent Antimonial Drug: Possible Role of Residual Sb(III) and Protective Effect of Ascorbic Acid

    PubMed Central

    Kato, Kelly C.; Morais-Teixeira, Eliane; Reis, Priscila G.; Silva-Barcellos, Neila M.; Salaün, Pascal; Campos, Paula P.; Dias Corrêa-Junior, José; Rabello, Ana; Demicheli, Cynthia

    2014-01-01

    Pentavalent antimonial drugs such as meglumine antimoniate (Glucantime [Glu; Sanofi-Aventis, São Paulo, Brazil]) produce severe side effects, including cardiotoxicity and hepatotoxicity, during the treatment of leishmaniasis. We evaluated the role of residual Sb(III) in the hepatotoxicity of meglumine antimoniate, as well as the protective effect of the antioxidant ascorbic acid (AA) during antimonial chemotherapy in a murine model of visceral leishmaniasis. BALB/c mice infected with Leishmania infantum were treated intraperitoneally at 80 mg of Sb/kg/day with commercial meglumine antimoniate (Glu) or a synthetic meglumine antimoniate with lower Sb(III) level (MA), in association or not with AA (15 mg/kg/day), for a 20-day period. Control groups received saline or saline plus AA. Livers were evaluated for hepatocytes histological alterations, peroxidase activity, and apoptosis. Increased proportions of swollen and apoptotic hepatocytes were observed in animals treated with Glu compared to animals treated with saline or MA. The peroxidase activity was also enhanced in the liver of animals that received Glu. Cotreatment with AA reduced the extent of histological changes, the apoptotic index, and the peroxidase activity to levels corresponding to the control group. Moreover, the association with AA did not affect the hepatic uptake of Sb and the ability of Glu to reduce the liver and spleen parasite loads in infected mice. In conclusion, our data supports the use of pentavalent antimonials with low residue of Sb(III) and the association of pentavalent antimonials with AA, as effective strategies to reduce side effects in antimonial therapy. PMID:24189251

  16. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  17. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  18. Comparison of short-term effects of insulin and essential fatty acids on the slowed nerve conduction of streptozotocin diabetes in rats.

    PubMed

    Julu, P O; Mutamba, A

    1991-11-01

    Early effects of insulin and essential fatty acids on nerve conduction were studied. Insulin-dependent diabetes was induced in rats using streptozocin (65 mg/kg, i.p.); control rats were treated with buffer. Five weeks later, diabetic rats were divided into 5 groups. Two groups were given oral essential fatty acids (75% linoleic and 9% gamma-linolenic acids) for a further 3 and 5 days, respectively. Two other groups received subcutaneous insulin for a further 3 or 5 days. A group of diabetic rats were left without further treatment. Motor nerve conduction velocity was measured terminally in all rats by stimulating the sciatic nerve and recording EMGs in the gastrocnemius muscle under urethane anaesthesia. Sensory nerve conduction velocity was measured by stimulating and recording from the saphenous nerve trunk. Diabetic rats had significantly slowed motor and sensory nerve conduction velocities after 5 weeks (16.7%, P less than 0.001). Three days treatment with either insulin or fatty acids corrected the slowed motor nerve conduction velocity to a normal level. Conduction velocity in myelinated sensory nerves was still 10% slower in diabetic rats treated with insulin for 3 days (P less than 0.01). It was above the control level by 11% in diabetic rats treated with fatty acids for the same period (P less than 0.01). Conduction velocities in both sensory and motor nerves were normal in diabetic rats treated with either insulin or fatty acids for 5 days. It was concluded that both insulin and essential fatty acids had early effects on nerve conduction in diabetic rats. The speed of their actions, and the magnitudes of responses were different in sensory and motor nerves. PMID:1663993

  19. ASCORBIC ACID TREATMENT TO REDUCE RESIDUAL HALOGEN-BASED OXIDANTS PRIOR TO THE DETERMINATION OF HALOGENATED DISINFECTION BYPRODUCTS IN POTABLE WATER

    EPA Science Inventory

    Treatment of potable water samples with ascorbic acid has been investigated as a means for reducing residual halogen-based oxidants (disinfectants)i.e., HOCl, Cl2, Brw and BrCl, prior to determination of EPA Method 551.1A and 551.1B analytes. These disinfection byproducts include...

  20. Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced soli...

  1. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

    PubMed Central

    Sundin, G W; Shankar, S; Chakrabarty, A M

    1996-01-01

    We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as

  2. Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity*

    PubMed Central

    Higashi, Kyohei; Sakamaki, Yoshiharu; Herai, Emiko; Demizu, Risa; Uemura, Takeshi; Saroj, Sunil D.; Zenda, Risa; Terui, Yusuke; Nishimura, Kazuhiro; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2010-01-01

    Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp8, Tyr43, Trp100, Leu110, and Tyr261 in PotB and Trp46, Asp108, Glu169, Ser196, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake and that Tyr160, Glu172, and Leu274 in PotB and Tyr19, Tyr88, Tyr148, Glu160, Leu195, and Tyr211 in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp8 in PotB was important for insertion of PotB and PotC into membranes. Tyr43, Trp100, and Leu110 in PotB and Trp46, Asp108, Ser196, and Asp198 in PotC were found to be involved in the interaction with PotD. Leu110 and Tyr261 in PotB and Asp108, Asp198, and Asp199 in PotC were involved in the recognition of spermidine, and Trp100 and Tyr261 in PotB and Asp108, Glu169, and Asp198 in PotC were involved in ATPase activity of PotA. Accordingly, Trp100 in PotB was involved in both PotD recognition and ATPase activity, Leu110 in PotB was involved in both PotD and spermidine recognition, and Tyr261 in PotB was involved in both spermidine recognition and ATPase activity. Asp108 and Asp198 in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity. PMID:20937813

  3. Correlation of deglutition in subacute ischemic stroke patients with peripheral blood adaptive immunity: Essential amino acid improvement.

    PubMed

    Aquilani, Roberto; Emilio, Benevolo; Dossena, Maurizia; Baiardi, Paola; Testa, Amidio; Boschi, Federica; Viglio, Simona; Iadarola, Paolo; Pasini, Evasio; Verri, Manuela

    2015-12-01

    We aimed to document in stroke patients peripheral blood immune cell profiles, their relations with neuro-functional tests, and any possible influence of supplemented essential amino acids (EAAs) may have on both the immune system and the relationship of the latter with neuro-function.Forty-two dysphagic stroke patients (27 men; 71±9 years) underwent bio-humoral measurements, neuro-functional tests, including Functional Independence Measure (FIM) and Dysphagia Outcome and Severity Scale (DOSS), and were randomized to receive EAAs 8 g/d (EAA group) or isocaloric maltodextrin (placebo group).At discharge all measurements were repeated 38±1 days after randomization.At admission, total white cell (TWC), neutrophil (N), and lymphocyte (Lymph) counts were normal and the N/Lymph ratio was higher than normal values (<3.0). At discharge, both TWC and N decreased while Lymph increased significantly. As a result, the N/Lymph ratio significantly decreased (P <0.001) returning to normal levels. Absolute Lymph counts and Lymph % TWC correlated positively with DOSS (r = +0.235, P = 0.04 and r = +0.224, P = 0.05, respectively), negatively with C-reactive protein natural logarithm (ln CRP) (P = 0.02 and P = 0.0001, respectively), which is an inflammation marker. N correlated positively with ln CRP (P = 0.001) and had a slight negative association with FIM (P = 0.07). The N/Lymph ratio was inversely related to FIM (r = -0.262, P = 0.02) and DOSS (r = -0.279, P = 0.01). Finally, FIM correlated with DOSS (r = +0.35, P = 0.05).For the regression analysis, the overtime changes of Lymph % TWC correlated significantly with DOSS (P = 0.01). There was a positive correlation between Lymph % TWC and DOSS for the entire stroke population (P = 0.015). While this correlation was not important for the placebo group (P = 0.27), it was significant in the EAA subgroup (P = 0.018).In the sub-acute stroke stage, there may be slight alterations of peripheral blood immune cells. Lymph cells are

  4. Interaction of acid mine drainage with Ordinary Portland Cement blended solid residues generated from active treatment of acid mine drainage with coal fly ash.

    PubMed

    Gitari, Wilson M; Petrik, Leslie F; Key, David L; Okujeni, Charles

    2011-01-01

    Fly ash (FA) has been investigated as a possible treatment agent for Acid mine drainage (AMD) and established to be an alternative, cheap and economically viable agent compared to the conventional alkaline agents. However, this treatment option also leads to generation of solid residues (SR) that require disposal and one of the proposed disposal method is a backfill in coal mine voids. In this study, the interaction of the SR with AMD that is likely to be present in such backfill scenario was simulated by draining columns packed with SR and SR + 6% Ordinary Portland Cement (OPC) unsaturated with simulated AMD over a 6 month period. The evolving geochemistry of the liquid/solid (L/S) system was evaluated in-terms of the mineral phases likely or controlling contaminants attenuation at the different pH regimes generated. Stepwise acidification of the percolates was observed as the drainage progressed. Two pH buffer zones were observed (7.5-9 and 3-4) for SR and (11.2-11.3 and 3.5-4) for SR + 6% OPC. The solid residue cores (SR) appeared to have a significant buffering capacity, maintaining a neutral to slightly alkaline pH in the leachates for an extended period of time (97 days: L/S 4.3) while SR + 6% OPC reduced this neutralization capacity to 22 days (L/S 1.9). Interaction of AMD with SR or SR + 6% OPC generated alkaline conditions that favored precipitation of Fe, Al, Mn-(oxy) hydroxides, Fe and Ca-Al hydroxysulphates that greatly contributed to the contaminants removal. However, precipitation of these phases was restricted to the pH of the leachates remaining at neutral to circum-neutral levels. Backfill of mine voids with SR promises to be a feasible technology for the disposal of the SR but its success will greatly depend on the disposal scenario, AMD generated and the alkalinity generating potential of the SR. A disadvantage would be the possible re-dissolution of the precipitated phases at pH < 4 that would release the contaminants back to the water column

  5. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr.

    PubMed

    Singh, S P; Tomkowicz, B; Lai, D; Cartas, M; Mahalingam, S; Kalyanaraman, V S; Murali, R; Srinivasan, A

    2000-11-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization

  6. Functional Role of Residues Corresponding to Helical Domain II (Amino Acids 35 to 46) of Human Immunodeficiency Virus Type 1 Vpr

    PubMed Central

    Singh, Satya P.; Tomkowicz, Brian; Lai, Derhsing; Cartas, Maria; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.; Murali, Ramachandran; Srinivasan, Alagarsamy

    2000-01-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G2, nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLΔVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprΔ35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike

  7. Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

    PubMed

    Worst, Emanuel G; Exner, Matthias P; De Simone, Alessandro; Schenkelberger, Marc; Noireaux, Vincent; Budisa, Nediljko; Ott, Albrecht

    2016-01-01

    The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any

  8. A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: Identification and mutagenesis of two conserved residues that are essential for enzyme activity

    SciTech Connect

    Wilks, H.M.; Timko, M.P.

    1995-01-31

    Protochlorophyllide reductase (NADPH:protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway. We have developed an expression system in which the protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme. By protein sequence comparisons, we have identified the plant protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases. Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity. A model of the enzyme reaction mechanism for light-dependent protochlorophyllide reduction is proposed. 33 refs., 5 figs.

  9. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cyt