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Sample records for acid residues essential

  1. Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

    PubMed

    Britten, C J; Bird, M I

    1997-02-11

    The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.

  2. 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.

  3. 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

  4. Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation.

    PubMed

    Deng, Z; Chen, C J; Zerby, D; Delecluse, H J; Lieberman, P M

    2001-11-01

    Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

  5. 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.

  6. 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

  7. 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.

  8. Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis.

    PubMed

    Li, Shu-Fen; Song, Li-Ying; Zhang, Guo-Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min

    2011-12-01

    In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.

  9. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity.

    PubMed

    Watanabe, T; Kobori, K; Miyashita, K; Fujii, T; Sakai, H; Uchida, M; Tanaka, H

    1993-09-01

    Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120.

    PubMed

    Zhang, Juan; Sun, Ya Fang; Zhao, Kai Hong; Zhou, Ming

    2012-12-10

    The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1. PMID:22982227

  15. Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation.

    PubMed

    Kihara, Akio; Sakuraba, Hiroko; Ikeda, Mika; Denpoh, Aki; Igarashi, Yasuyuki

    2008-04-25

    Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

  16. 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

  17. Mutagenesis of essential functional residues in acetylcholinesterase.

    PubMed Central

    Gibney, G; Camp, S; Dionne, M; MacPhee-Quigley, K; Taylor, P

    1990-01-01

    The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis. Images PMID:2217185

  18. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  19. An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1.

    PubMed

    Ben-Yona, Assaf; Kanner, Baruch I

    2012-03-01

    GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.

  20. Consequences of Essential Fatty Acids

    PubMed Central

    Lands, Bill

    2012-01-01

    Essential fatty acids (EFA) are nutrients that form an amazingly large array of bioactive mediators that act on a large family of selective receptors. Nearly every cell and tissue in the human body expresses at least one of these receptors, allowing EFA-based signaling to influence nearly every aspect of human physiology. In this way, the health consequences of specific gene-environment interactions with these nutrients are more extensive than often recognized. The metabolic transformations have similar competitive dynamics for the n-3 and n-6 homologs when converting dietary EFA from the external environment of foods into the highly unsaturated fatty acid (HUFA) esters that accumulate in the internal environment of cells and tissues. In contrast, the formation and action of bioactive mediators during tissue responses to stimuli tend to selectively create more intense consequences for n-6 than n-3 homologs. Both n-3 and n-6 nutrients have beneficial actions, but many common health disorders are undesired consequences of excessive actions of tissue n-6 HUFA which are preventable. This review considers the possibility of preventing imbalances in dietary n-3 and n-6 nutrients with informed voluntary food choices. That action may prevent the unintended consequences that come from eating imbalanced diets which support excessive chronic actions of n-6 mediators that harm human health. The consequences from preventing n-3 and n-6 nutrient imbalances on a nationwide scale may be very large, and they need careful evaluation and implementation to avoid further harmful consequences for the national economy. PMID:23112921

  1. 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 Central

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    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

  2. 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

  3. Srchi24, A Chitinase Homolog Lacking an Essential Glutamic Acid Residue for Hydrolytic Activity, Is Induced during Nodule Development on Sesbania rostrata1

    PubMed Central

    Goormachtig, Sofie; Van de Velde, Willem; Lievens, Sam; Verplancke, Christa; Herman, Sylvia; De Keyser, Annick; Holsters, Marcelle

    2001-01-01

    The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots. Stem nodulation was used as a model system to isolate early markers by differential display. One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation. This enhancement depended on Nod factor-producing bacteria. Srchi24 transcript levels were induced also by exogenous cytokinins. In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules. Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue. A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity. A function in nodulation is proposed for the Srchi24 protein. PMID:11553736

  4. 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

  5. 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.

  6. 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

  7. [Essential fatty acids and the skin].

    PubMed

    Berbis, P; Hesse, S; Privat, Y

    1990-06-01

    Metabolism of the essential fatty acids (AGE) in an organism leads to synthesis of eicosanoids, which have various biological properties. Linoleic acid plays an important part in maintenance of epidermal integrity by intervening in the cohesion of the stratum corneum and in prevention of transepidermal water loss. Metabolites of arachidonic acid (mostly those obtained by the lipoxygenase pathway) are important agents in causing many inflammatory skin reactions concurrent with development of skin diseases such as psoriasis and atopic dermatitis. Pharmacological and dietetic control of the metabolism of arachidonic acid is a new and interesting therapeutic concept in the care of skin diseases. Also, fish oil, which is rich in linoleic acid and poor in arachidonic acid, seems to be useful in basal treatment of psoriasis. The value of evening primrose oil, which is rich in gamma-linoleic acid, in the treatment of atopic dermatitis is discussed.

  8. Essential fatty acid deficiency in malnourished children.

    PubMed

    Holman, R T; Johnson, S B; Mercuri, O; Itarte, H J; Rodrigo, M A; De Tomas, M E

    1981-08-01

    Fatty acid patterns of major classes of lipids of serum were measured in forty Argentine children ages 2 to 24 months admitted to the hospital with chronic malnutrition. A normal control group of 48 children from the same population was also examined. Serum lipids were extracted and separated into phospholipids, cholesteryl esters, triglycerides, and free fatty acids. These were converted to methyl esters which were analyzed by gas chromatography. In chronic malnutrition, the fatty acid patterns of phospholipids and cholesteryl esters indicated changes characteristic of essential fatty acid deficiency of moderate degree. The total omega 6 acids were found to be highly significantly diminished from normal, and the ratio of 20:3 omega 9/20:4 omega 6 was highly significantly increased. Decreased proportions of omega 6 metabolites suggested impaired desaturase activity, and elevated ratios of 22:4 omega 6/20:4 omega 6 and 20:2 omega 6/18:2 omega 6 suggested increased chain elongation in chronic malnutrition.

  9. Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

    PubMed

    Batt, C A; Jamieson, A C; Vandeyar, M A

    1990-01-01

    Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. PMID:2405386

  10. Essential fatty acids and human brain.

    PubMed

    Chang, Chia-Yu; Ke, Der-Shin; Chen, Jen-Yin

    2009-12-01

    The human brain is nearly 60 percent fat. We've learned in recent years that fatty acids are among the most crucial molecules that determine your brain's integrity and ability to perform. Essential fatty acids (EFAs) are required for maintenance of optimal health but they can not synthesized by the body and must be obtained from dietary sources. Clinical observation studies has related imbalance dietary intake of fatty acids to impaired brain performance and diseases. Most of the brain growth is completed by 5-6 years of age. The EFAs, particularly the omega-3 fatty acids, are important for brain development during both the fetal and postnatal period. Dietary decosahexaenoic acid (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. Beyond their important role in building the brain structure, EFAs, as messengers, are involved in the synthesis and functions of brain neurotransmitters, and in the molecules of the immune system. Neuronal membranes contain phospholipid pools that are the reservoirs for the synthesis of specific lipid messengers on neuronal stimulation or injury. These messengers in turn participate in signaling cascades that can either promote neuronal injury or neuroprotection. The goal of this review is to give a new understanding of how EFAs determine our brain's integrity and performance, and to recall the neuropsychiatric disorders that may be influenced by them. As we further unlock the mystery of how fatty acids affect the brain and better understand the brain's critical dependence on specific EFAs, correct intake of the appropriate diet or supplements becomes one of the tasks we undertake in pursuit of optimal wellness.

  11. DOCOSAHEXAENOIC ACID AND ARACHIDONIC ACID PREVENT ESSENTIAL FATTY ACID DEFICIENCY AND HEPATIC STEATOSIS

    PubMed Central

    Le, Hau D.; Meisel, Jonathan A.; de Meijer, Vincent E.; Fallon, Erica M.; Gura, Kathleen M.; Nose, Vania; Bistrian, Bruce R.; Puder, Mark

    2012-01-01

    Objectives Essential fatty acids are important for growth, development, and physiologic function. Alpha-linolenic acid and linoleic acid are the precursors of docosahexaenoic and arachidonic acid, respectively, and have traditionally been considered the essential fatty acids. However, we hypothesized that docosahexaenoic acid and arachidonic acid can function as the essential fatty acids. Methods Using a murine model of essential fatty acid deficiency and consequent hepatic steatosis, we provided mice with varying amounts of docosahexaenoic and arachidonic acids to determine whether exclusive supplementation of docosahexaenoic and arachidonic acids could prevent essential fatty acid deficiency and inhibit or attenuate hepatic steatosis. Results Mice supplemented with docosahexaenoic and arachidonic acids at 2.1% or 4.2% of their calories for 19 days had normal liver histology and no biochemical evidence of essential fatty acid deficiency, which persisted when observed after 9 weeks. Conclusion Supplementation of sufficient amounts of docosahexaenoic and arachidonic acids alone without alpha-linolenic and linoleic acids meets essential fatty acid requirements and prevents hepatic steatosis in a murine model. PMID:22038210

  12. 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

  13. Essential fatty acid requirements of cats: pathology of essential fatty acid deficiency.

    PubMed

    MacDonald, M L; Anderson, B C; Rogers, Q R; Buffington, C A; Morris, J G

    1984-07-01

    The pathologic changes of essential fatty acid (EFA) deficiency were studied in specific-pathogen-free, domestic shorthair cats which were fed purified diets for 1.5 to 2.5 years. Cats fed an EFA-deficient diet exhibited signs of deficiency: severe fatty degeneration of the liver, excessive fat in the kidneys, dystrophic mineralization of the adrenal glands, degeneration of the testes, and hyperkeratosis of the skin. Minor clinical pathologic changes were consistent with liver damage. Fatty acid analyses of plasma lipids revealed low concentrations of linoleate and other n6-fatty acids, and high concentrations of n7- and n9-fatty acids, consistent with EFA deficiency. These signs of deficiency were prevented by including safflower seed oil in the diet at a concentration to supply linoleate at 6.7% of dietary energy. Therefore, linoleate is an EFA for the cat, despite negligible conversion of linoleate to arachidonate in cat liver. However, in cats fed a diet containing linoleate, but lacking arachidonate, there was mild mineralization of the kidneys, and the neutral fat content of the liver was slightly higher than that of cats fed a diet containing arachidonate and other long-chain polyunsaturated fatty acids. Also, 2 of the 19 cats fed arachidonate-deficient diets developed unusual inflammatory skin lesions. In cats fed a diet containing hydrogenated coconut oil, safflower seed oil, and chicken fat, fatty livers developed despite the presence of high levels of linoleate. The fatty livers appeared to result from a specific deleterious effect of the medium-chain triglycerides in hydrogenated coconut oil. Most of the organ pathologic changes of EFA deficiency in the cat can be prevented by feeding dietary linoleate. Linoleate meets the EFA requirement for functions which depend on proper membrane structure: growth, lipid transport, normal skin and coat condition, and maintenance of the epidermal permeability barrier. However, dietary arachidonate is required by the

  14. Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.

    PubMed

    Asenjo, Ana; Mendieta, Jesús; Gómez-Puertas, Paulino; Villanueva, Nieves

    2008-03-01

    Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Rats rapidly reject diets deficient in essential amino acids.

    PubMed

    Koehnle, Thomas J; Russell, Matthew C; Gietzen, Dorothy W

    2003-07-01

    Omnivores must obtain diets balanced with respect to amino acids to support growth and protein synthesis. The standard paradigm used to study behavioral responses to amino acid deficiency combines deficient diets with dietary novelty. The objective of this study was to examine the effects of amino acid deficiency on the first meal of rats without the confounding effects of novelty. We report on a series of five studies of feeding behavior in rats. Rats were fed low protein diets for 5-7 d and then exposed to diets with and without essential amino acids. Rats consistently demonstrated recognition of essential amino acid deficiency within the first meal by a significant reduction in first meal duration, rejecting the deficient diets after just 12-16 min exposure. This is the first report of a rapid effect of amino acid-deficient diets without the confounding effects of dietary novelty.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. Biosynthesis of 'essential' amino acids by scleractinian corals.

    PubMed Central

    Fitzgerald, L M; Szmant, A M

    1997-01-01

    Animals rely on their diet for amino acids that they are incapable either of synthesizing or of synthesizing in sufficient quantities to meet metabolic needs. These are the so-called 'essential amino acids'. This set of amino acids is similar among the vertebrates and many of the invertebrates. Previously, no information was available for amino acid synthesis by the most primitive invertebrates, the Cnidaria. The purpose of this study was to examine amino acid synthesis by representative cnidarians within the Order Scleractinia. Three species of zooxanthellate reef coral, Montastraea faveolata, Acropora cervicornis and Porites divaricata, and two species of non-zooxanthellate coral, Tubastrea coccinea and Astrangia poculata, were incubated with 14C-labelled glucose or with the 14C-labelled amino acids glutamic acid, lysine or valine. Radiolabel tracer was followed into protein amino acids. A total of 17 amino acids, including hydroxyproline, were distinguishable by the techniques used. Of these, only threonine was not found radiolabelled in any of the samples. We could not detect tryptophan or cysteine, nor distinguish between the amino acid pairs glutamic acid and glutamine, or aspartic acid and asparagine. Eight amino acids normally considered essential for animals were made by the five corals tested, although some of them were made only in small quantities. These eight amino acids are valine, isoleucine, leucine, tyrosine, phenylalanine histidine, methionine and lysine. The ability of cnidarians to synthesize these amino acids could be yet another indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa. PMID:9078264

  5. 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.

  6. 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

  7. 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.

  8. A single valine residue plays an essential role in peripherin/rds targeting to photoreceptor outer segments.

    PubMed

    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.

  9. Ligatin binds phosphohexose residues on acidic hydrolases.

    PubMed

    Jakoi, E R; Kempe, K; Gaston, S M

    1981-01-01

    Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N-acetyl beta-D-glucosaminidase (beta-NAG) were solubilized with this receptor. The solubilized beta-NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. beta-N-Acetyl D-glucosaminidase-ligatin complexes were dissociated by low concentrations of mannose 6-phosphate (Man6P) and/or glucose 1-phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal beta-NAG-ligatin complexes showed a four-fold preferential dissociation with Man6P; macrophage complexes showed a 160-fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous-32 label from these enzymes and prevented binding of beta-NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of beta-NAG to ligatin by no more than 30%. This apparent resistance of beta-NAG to dephosphorylation was consistent with the chromatographic behavior of QAE of 3H-labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on beta-NAG analogous to the NAC glucosamine 1 P6 mannose present on beta-glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfield, S: J Biol Chem 255: 6633, 1980). PMID:7299841

  10. Stable nitrogen isotopes in essential versus non-essential amino acids of different plankton size fractions.

    PubMed

    Loick, Natalie; Gehre, Matthias; Voss, Maren

    2007-12-01

    The stable nitrogen isotope values (delta(15)N) of the essential amino acid (EAA) leucine and the delta(15)N values of six non-essential amino acids (NEAAs) from plankton size fractions from the South China Sea (SCS) were analysed. Data from the SCS were collected during two cruises in July 2003 and 2004 onboard of RV Nghien Cuu Bien. The delta(15)N values of alanine, aspartic acid, glutamic acid and leucine increased with size at all sites. The delta(15)N of glycine did not increase with size, the delta(15)N of tyrosine increased with size only at offshore stations and the delta(15)N of proline increased with size only at inshore stations. We found highly significant correlations between the delta(15)N ratios of leucine to the delta(15)N ratios of glutamic acid, proline, alanine, tyrosine and aspartic acid at oligotrophic sites of enhanced nitrogen fixation. In contrast thereto these correlations were less distinct or absent at more eutrophic sites of low nitrogen fixation. A comparison with an independent data set from the tropical North Atlantic revealed intriguing similar patterns. We interpret these patterns as result of the connected metabolism of EAA and NEAA in zooplankton at sites of nitrogen limitation.

  11. 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.

  12. 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.

  13. Electron microscopy of carbonaceous matter in Allende acid residues

    NASA Astrophysics Data System (ADS)

    Lumpkin, G. R.

    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.

  14. 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.

  15. Essential fatty acids and sleep: mini-review and hypothesis.

    PubMed

    Yehuda, S; Rabinovitz, S; Mostofsky, D I

    1998-02-01

    The neurochemical basis of sleep mechanisms (onset and maintenance) is still controversial although the phenomenon itself is known to be mediated by more than a single molecule. The list of suggested endogenous sleep substances is rather long, and there is no single 'sleep center' identified in the brain. The role of fatty acids, and essential fatty acids in particular, has been ignored in sleep research. This review proposes an integration of the current knowledge about the effects of fatty acids in sleep neurochemistry, wherein fatty acids are seen to exert a direct effect on neuronal membrane structure or indirectly on the dynamics of biochemical compounds (complex lipids, prostaglandins, neurotransmitters, amino acids, interleukins) necessary for the initiation and maintenance of sleep.

  16. Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity.

    PubMed

    Wen, L; Miao, Z W; Qing, W D

    1999-08-01

    The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine. PMID:10609644

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein.

    PubMed

    Bhubhanil, Sakkarin; Ruangkiattikul, Nantaporn; Niamyim, Phettree; Chamsing, Jareeya; Ngok-Ngam, Patchara; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2012-10-01

    The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant. PMID:22817265

  8. A color-determining amino acid residue of proteorhodopsin.

    PubMed

    Ozaki, Yuya; Kawashima, Takayoshi; Abe-Yoshizumi, Rei; Kandori, Hideki

    2014-09-30

    Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. More than 1000 PRs are classified as blue-absorbing (λmax ∼ 490 nm) and green-absorbing (λmax ∼ 525 nm) PRs. The color determinant is known to be at position 105, where blue-absorbing and green-absorbing PRs possess Gln and Leu, respectively. This suggests hydrophobicity at position 105 plays a key role in color tuning. Here we successfully introduced 19 amino acid residues into position 105 of green-absorbing PR in the membrane environment and investigated the absorption properties. High-performance liquid chromatography analysis shows that the isomeric composition of the all-trans form is >70% for all mutants, indicating little influence of different isomers on color tuning. Absorption spectra of the wild-type and 19 mutant proteins were well-characterized by the pH-dependent equilibria of the protonated and deprotonated counterion (Asp97) of the Schiff base, whereas the λmax values of these two states and the pKa value differed significantly among mutants. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, the λmax values of the two states and the pKa value did not correlate with the hydropathy index of residues. In contrast, the λmax and pKa were correlated with the volume of residues, though Gln and Leu possess similar volumes. This observation concludes that the λmax and pKa of Asp97 are determined by local and specific interactions in the Schiff base moiety, in which the volume of the residue at position 105 is more influential than its hydrophobicity. We suggest that the hydrogen-bonding network in the Schiff base moiety plays a key role in the λmax and pKa of Asp97, and the hydrogen-bonding network is significantly perturbed by large amino acid residues but may be preserved by additional water molecule(s) for small amino acid residues at position 105. PMID:25180875

  9. 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

    2016-07-07

    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.

  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. 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

  12. 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.

  13. Essential, completely conserved glycine residue in the domain III S2-S3 linker of voltage-gated calcium channel alpha1 subunits in yeast and mammals.

    PubMed

    Iida, Kazuko; Teng, Jinfeng; Tada, Tomoko; Saka, Ayaka; Tamai, Masumi; Izumi-Nakaseko, Hiroko; Adachi-Akahane, Satomi; Iida, Hidetoshi

    2007-08-31

    Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.

  14. Amino acid residues modulating the activities of staphylococcal glutamyl endopeptidases.

    PubMed

    Ono, Toshio; Ohara-Nemoto, Yuko; Shimoyama, Yu; Okawara, Hisami; Kobayakawa, Takeshi; Baba, Tomomi T; Kimura, Shigenobu; Nemoto, Takayuki K

    2010-10-01

    The glutamyl endopeptidase family of enzymes from staphylococci has been shown to be important virulence determinants of pathogenic family members, such as Staphylococcus aureus. Previous studies have identified the N-terminus and residues from positions 185-195 as potentially important regions that determine the activity of three members of the family. Cloning and sequencing of the new family members from Staphylococcus caprae (GluScpr) and Staphylococcus cohnii (GluScoh) revealed that the N-terminal Val residue is maintained in all family members. Mutants of the GluV8 enzyme from S. aureus with altered N-terminal residues, including amino acids with similar properties, were inactive, indicating that the Val residue is specifically required at the N-terminus of this enzyme family in order for them to function correctly. Recombinant GluScpr was found to have peptidase activity intermediate between GluV8 and GluSE from Staphylococcus epidermis and to be somewhat less specific in its substrate requirements than other family members. The 185-195 region was found to contribute to the activity of GluScpr, although other regions of the enzyme must also play a role in defining the activity. Our results strongly indicate the importance of the N-terminal and the 185-195 region in the activity of the glutamyl endopeptidases of staphylococci. PMID:20707600

  15. 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

  16. 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.

  17. Involvement of Acidic Amino Acid Residues in Zn(2+) Binding to Respiratory Complex I.

    PubMed

    Kriegel, Sébastien; Srour, Batoul; Steimle, Stefan; Friedrich, Thorsten; Hellwig, Petra

    2015-09-21

    Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm(-1)) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn(2+) binding. In variant D563N(L), which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn(2+) binding is changed. Our data support a role for this residue in proton translocation.

  18. Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

    PubMed Central

    Reguera, Juan; Carreira, Aura; Riolobos, Laura; Almendral, José María; Mateu, Mauricio G.

    2004-01-01

    Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles. PMID:14981262

  19. 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

  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. Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy.

    PubMed

    Zhao, Bo; Zhang, Qi

    2015-10-28

    Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states--or excited conformational states--that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond (13)C-(1)H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.

  2. 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.

  3. 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

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

    PubMed

    Tessari, Paolo; Lante, Anna; Mosca, Giuliano

    2016-05-25

    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.

  5. 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

  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. 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

  8. 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.

  9. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Tolerances § 180.155 1-Naphthaleneacetic acid; tolerances for residues. (a) General. Tolerances are established for the combined residues of the plant growth regulator 1-naphthaleneacetic acid and its... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 1-Naphthaleneacetic acid;...

  10. 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

  11. 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

  12. A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2014-11-01

    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

  13. 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

  14. Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

    PubMed Central

    Sigal, C T; Zhou, W; Buser, C A; McLaughlin, S; Resh, M D

    1994-01-01

    Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation. Images PMID:7527558

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

    PubMed

    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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

    PubMed Central

    Sandy, James; Mushtaq, Adeel; Holton, Simon J.; Schartau, Pamela; Noble, Martin E. M.; Sim, Edith

    2005-01-01

    The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity. PMID:15869465

  1. A critical amino acid residue, asp446, in UDP-glucuronosyltransferase.

    PubMed Central

    Iwano, H; Yokota, H; Ohgiya, S; Yotumoto, N; Yuasa, A

    1997-01-01

    An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from YshA1337GG1384A had no transferase activity. Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme. PMID:9271076

  2. Evaluation of residues of essential oil components in honey after different anti-varroa treatments.

    PubMed

    Adamczyk, Sabine; Lázaro, Regina; Pérez-Arquillué, Consuelo; Conchello, Pilar; Herrera, Antonio

    2005-12-28

    Apiary trials on the use of three different treatments (Apilife Var, thymol solution in olive oil, and thymol solution in ethanol) for the control of Varroa destructor were conducted in Aragon (northeastern Spain). For the evaluation of the presence of residues of these treatments in honey an analytical method was developed. The method is applied to analyze honey samples before and after treatments with the acaricides mentioned. A solid-phase extraction on trifunctional silane SPE C18 cartridge and gas chromatography separation using a flame ionization detector allow reliable and precise determination of residues of thymol, menthol, eucalyptol, and camphor in honey. The results indicate that camphor is present in only low concentrations, residues of eucalyptol or menthol were not found at all, and only thymol left residues in high concentrations. Residues of thymol found in honey collected from the beehives ranged from 0.75 to 8.20 microg/g for Apilife Var, from 0.03 to 6.30 microg/g for thymol solution in olive oil, and from 0.05 to 6.20 microg/g for thymol solution in ethanol. Even so, natural treatments can be considered to be good alternatives for synthetic acaricides, especially because they do not represent a sanitary risk. PMID:16366699

  3. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)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...

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)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...

  5. 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...

  6. 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

  7. 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

  8. 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

  9. The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

    PubMed Central

    Kleifeld, Oded; Shi, Shu Ping; Zarivach, Raz; Eisenstein, Miriam; Sagi, Irit

    2003-01-01

    Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover. PMID:12592017

  10. 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.

  11. 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

  12. Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough).

    PubMed

    Zhou, Z; Swenson, R P

    1995-03-14

    The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family

  13. 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

  14. Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality.

    PubMed

    Galili, Gad; Amir, Rachel

    2013-02-01

    Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.

  15. None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

    PubMed Central

    Chiwata, Ryohei; Kohori, Ayako; Kawakami, Tomonari; Shiroguchi, Katsuyuki; Furuike, Shou; Adachi, Kengo; Sutoh, Kazuo; Yoshida, Masasuke; Kinosita, Kazuhiko

    2014-01-01

    F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions. PMID:24853745

  16. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    PubMed

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  17. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  18. 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...

  19. 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...

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  1. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  2. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  3. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  4. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  5. Identification of Conformationally Sensitive Residues Essential for Inhibition of Vesicular Monoamine Transport by the Noncompetitive Inhibitor Tetrabenazine*

    PubMed Central

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

    2013-01-01

    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

  6. Identification of essential residues involved in the glutamate binding pocket of the group II metabotropic glutamate receptor.

    PubMed

    Malherbe, P; Knoflach, F; Broger, C; Ohresser, S; Kratzeisen, C; Adam, G; Stadler, H; Kemp, J A; Mutel, V

    2001-11-01

    Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled receptors that play central roles as modulators of both glutamatergic and other major neurotransmitter systems in CNS. Using molecular modeling, site-directed mutagenesis, [(3)H]LY354740 binding, [(35)S]GTPgammaS binding, and activation of GIRK current, we have been able to identify residues crucial for the binding of LY354740 and glutamate to rat mGlu2 receptors. Several of the crucial residues located in the binding site (Arg-57, Tyr-144, Tyr-216, Asp-295) have not been identified previously. We propose that the gamma-carboxyl group of LY354740 forms H-bonds to Arg-57, whereas the alpha-carboxyl group forms an H-bond with the hydroxyl group of Ser-145. The alpha-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl group of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/pi)-interaction with the bicyclo-hexane ring of LY354740. Furthermore, the mutation of residues Ser-148 and Arg-183, which are too remote for a direct interaction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are involved in closure of the domain. Finally, Asp-146, which is also remote from the binding site, was shown to be involved in the differential binding affinity of [(3)H]LY354740 for mGlu2 versus mGlu3 receptors. All the mGlu receptors except mGlu2 are activated by Ca(2+) and have serine instead of aspartic acid at this position, which suggests a critical role of this aspartic acid residue in the binding properties of this unique receptor. PMID:11641422

  7. 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…

  8. Esterification of essential and non-essential fatty acids into distinct lipid classes in ruminant and non-ruminant tissues.

    PubMed

    Caldari-Torres, Cristina; McGilliard, Michael L; Corl, Benjamin A

    2016-10-01

    Extensive microbial biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen reduces the essential fatty acids (EFA) available for absorption in ruminant animals, but there is no published documentation of ruminants developing EFA deficiency. In ruminants, most circulating PUFA are found in the phospholipid (PL) and cholesteryl ester lipid classes that have slow turn-over compared to other lipid classes. The objective of this experiment was to measure fatty acid esterification patterns of the non-EFA palmitic (16:0) and oleic acid (18:1), and the EFA linoleic (18:2) and linolenic acid (18:3) in small intestine, liver, and muscle tissue of cows and pigs to identify tissues participating in sequestration of these FA in less metabolically active lipid classes in ruminants. Bovine and porcine small intestine, liver, and muscle explants were prepared and incubated in media containing radiolabeled 16:0, 18:1, 18:2, or 18:3 to measure esterification of fatty acids into PL and TG. Neither bovine nor porcine small intestine explants preferentially incorporated non-EFA compared to EFA into PL vs TG. Bovine liver explants esterified a larger proportion of EFA than non-EFA into PL compared to TG, while incorporation was similar among the FA tested in porcine liver explants. Bovine muscle explants showed preferential incorporation of EFA into PL rather than TG. Results show that bovine and porcine liver and muscle esterify EFA and non-EFA differently and that the conservation of EFA in ruminants is a result of preferential incorporation of EFA into PL mediated by bovine liver and muscle, but not the small intestine. PMID:27134010

  9. Protective Effect of Ocimum basilicum Essential Oil Against Acetic Acid-Induced Colitis in Rats.

    PubMed

    Rashidian, Amir; Roohi, Parnia; Mehrzadi, Saeed; Ghannadi, Ali Reza; Minaiyan, Mohsen

    2016-10-01

    Ocimum basilicum L has been traditionally used for the treatment of inflammatory bowel disease in Iran. This study investigates the ameliorative effect of Ocimum basilicum essential oil on an acetic acid-induced colitis model in rats. Ocimum basilicum essential oil with 2 doses (200 and 400 μL/kg) significantly ameliorated wet weight/length ratio of colonic tissue compared to the control group. Higher doses of essential oil (200 and 400 μL/kg) significantly reduced ulcer severity, ulcer area, and ulcer index. On the other hand, histological examination revealed the diminution of total colitis index as a marker for inflammatory cell infiltration in the colonic segments of rats treated with Ocimum basilicum essential oil (200 and 400 μL/kg). The increased level of myeloperoxidase was significantly decreased after the treatment with the essential oil (200 and 400 μL/kg). These results suggest that Ocimum basilicum exhibits protective effect against acetic acid-induced colitis.

  10. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-01

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  11. [Oregano essential oil as an inhibitor of higher fatty acid oxidation].

    PubMed

    Terenina, M B; Misharina, T A; Krikunova, N I; Alinkina, E S; Fatkulina, L D; Vorob'eva, A K

    2011-01-01

    Inhibition of the oxidation of fatty acids methyl esters by oregano essential oil was studied using capillary gas chromatography. A mixture of fatty acids which contained saturated, mono-, di-, and polyunsaturated acids with 16-24 carbon atoms was extracted from mice brain. Changes in the composition of esters in hexane solutions both in the presence of oregano essential oil and without it were examined during their autooxidation in light for 1 year. It was found that the oxidation rate of unsaturated fatty acids increases with increasing degree of their unsaturation. Oregano essential oil inhibited the oxidation process. Antioxidant activity of the oil increased with increase of its concentration. It was shown that carvacrol and thymol are the main antioxidant components of oregano oil. PMID:21950127

  12. Core Amino Acid Residues in the Morphology-Regulating Protein, Mms6, for Intracellular Magnetite Biomineralization

    PubMed Central

    Yamagishi, Ayana; Narumiya, Kaori; Tanaka, Masayoshi; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology. PMID:27759096

  13. Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

    PubMed Central

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing

    2014-01-01

    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism. PMID:24895685

  14. A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria.

    PubMed

    Zhou, Junpei; Shi, Pengjun; Huang, Huoqing; Cao, Yanan; Meng, Kun; Yang, Peilong; Zhang, Rui; Chen, Xiaoyan; Yao, Bin

    2010-12-01

    The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-D-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).

  15. Amino acid network for prediction of catalytic residues in enzymes: a comparison survey.

    PubMed

    Zhou, Jianhong; Yan, Wenying; Hu, Guang; Shen, Bairong

    2016-01-01

    Catalytic residues play a significant role in enzyme functions. With the recent accumulation of experimentally determined enzyme 3D structures and network theory on protein structures, the prediction of catalytic residues by amino acid network (AAN, where nodes are residues and links are residue interactions) has gained much interest. Computational methods of identifying catalytic residues are traditionally divided into two groups: sequence-based and structure-based methods. Two new structure- based methods are proposed in current advances: AAN and Elastic Network Model (ENM) of enzyme structures. By concentrating on AAN-based approach, we herein summarized network properties for predictions of catalytic residues. AAN attributes were showed responsible for performance improvement, and therefore the combination of AAN with previous sequence and structural information will be a promising direction for further improvement. Advantages and limitations of AAN-based methods, future perspectives on the application of AAN to the study of protein structure-function relationships are discussed.

  16. 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.

  17. 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

  18. [Oxytetracycline and oxolinic acid residues in kuruma prawn (Penaeus japonicus) and the effect of cooking procedures on the residues].

    PubMed

    Uno, Kazuaki

    2002-04-01

    Tissue distribution and residue depletion of oxytetracycline (OTC) and oxolinic acid (OA) were studied in the kuruma prawn (Penaeus japonicus). The prawn were kept in tanks with recirculated artificial seawater at a salinity of 22-23@1000. The water temperature was maintained at 25 degrees C. The average body weight was 22.9 +/- 4.9 g for OTC and 22.5 +/- 3.6 g for OA. The drug was mixed with the diet and orally administered through a catheter to the prawn. The doses of OTC and OA, respectively, were 50 mg/kg body weight. At each sample time, four prawns were sacrificed and tissues were sampled. OTC and OA levels were determined by high-performance liquid chromatography. At the highest levels, the concentrations of OTC were in the other: shell (13.57 micrograms/g) > hemolymph (12.20 micrograms/mL) > muscle (8.30 micrograms/g). For OA, the order was: shell (20.74 micrograms/g) > hemolymph (7.06 micrograms/mL) > muscle (2.05 micrograms/g). The elimination half-lives of hemolymph and muscle were 44.7 and 46.8 hours for OTC and 55.0 and 107.9 hours for OA, respectively. Residual OTC could not be detected in hemolymph and muscle at 20 days after dosing. Residual OA disappeared from hemolymph and muscle at 25 days after dosing. A 25-day period for OTC and 30-day period for OA could be regarded as the proper withdrawal time established for kuruma prawn by the Pharmaceutical Law in Japan. However, the elimination half-lives of shell for OTC and OA could not be calculated because both drug residues persisted in shell tissues, and the elimination phase was not completed during the experimental period. Residual OTC (14.10 +/- 2.26 micrograms/g, n = 6) and OA (0.32 +/- 0.06 microgram/g, n = 7) were detected in exuviae at 3 days and 4 days after dosing, respectively. Residual OTC was reduced to 50-70% in muscle by the usual methods of cooking (boiling, baking at 200 degrees C and frying at 180 degrees C), whereas reduction levels in shell were only 20-30%. Residual OA was

  19. Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

    PubMed

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

    2015-05-01

    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.

  20. 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

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

    DOE PAGES

    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

  2. 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.

  3. Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

    PubMed Central

    Sagane, Koji; Umemura, Mariko; Ogawa-Mitsuhashi, Kaoru; Tsukahara, Kappei; Yoko-o, Takehiko; Jigami, Yoshifumi

    2011-01-01

    Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops. PMID:21367863

  4. Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability.

    PubMed

    Sun, Lichang; Zhou, Yan; Liu, Runxia; Li, Yanhua; Gao, Fei; Wang, Xiaomin; Fan, Hongjie; Yuan, Shishan; Wei, Zuzhang; Tong, Guangzhi

    2015-02-01

    ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.

  5. Studies of the effects of essential fatty acid deficiency in the rat.

    PubMed

    Cox, J W; Rutecki, G W; Francisco, L L; Ferris, T F

    1982-12-01

    We report a model of prostaglandin depletion induced in rats by fasting for 11 days, followed by institution of an essential fatty acid-deficient diet. Urinary prostaglandin E, 2 weeks after this diet had been implemented, was 22 +/- 2 ng/24 hours compared to 113 +/- 8.5 ng/24 hours in controls (P less than 0.01). There was no difference in 24-hour urine volume or solute excretion in controls and essential fatty acid-deficient rats. Five hours after administration of NaCl, 10 mM/kg, essential fatty acid-deficient diet rats excreted 1.85 +/- 0.78 ml urine compared to 6.42 +/- 2.26 ml in control (p less than 0.01) with Na+ excretion 447 +/- 273 muEq in essential fatty acid-deficient rats vs 1483 +/- 366 muEq in control (P less than 0.01). Intravenous isotonic NaCl, 1.5% body weight, resulted in increased urine flow rate in control rats from 8.3 +/- 2 microliter/min to 28.7 +/- 8.8 microliter/min with sodium excretion increasing from 0.19 +/- 0.2 to 3.3 +/- 0.9 muEq/min. In the essential fatty acid-deficient diet animals, there was no significant change in flow rate, 6.07 +/- 2.43 to 9.85 +/- 4.29 microliter/min, or sodium excretion, 0.09 +/- 0.03 to 0.40 +/- 0.24 muEq, after saline infusion. There was no difference in the glomerular filtration rate of plasma aldosterone in the two groups after the salt load. When given a water load, 3 ml/100 g body weight, essential fatty acid-deficient diet rats excreted 2.5 +/- 0.7 ml in 5 hours compared to 6.3 +/- 1.4 ml in controls (P less than 0.01). The defect in water excretion was not due to increased sensitivity to antidiuretic hormone, since similar sensitivity to this hormone was demonstrated in the essential fatty acid-deficient diet and control rats during a water diuresis. When isotonic saline was substituted for drinking water, there was an increase in systolic blood pressure in essential fatty acid-deficient diet rats from 124 +/- 2 to 142 +/- 3 mm Hg over 9 days (P less than 0.01) compared to 122 +/- 2 before and 122

  6. Dietary essential amino acids affect the reproduction of the keystone herbivore Daphnia pulex.

    PubMed

    Fink, Patrick; Pflitsch, Claudia; Marin, Kay

    2011-01-01

    Recent studies have indicated that nitrogen availability can be an important determinant of primary production in freshwater lakes and that herbivore growth can be limited by low dietary nitrogen availability. Furthermore, a lack of specific essential nitrogenous biochemicals (such as essential amino acids) might be another important constraint on the fitness of consumers. This might be of particular importance for cladoceran zooplankton, which can switch between two alternative reproductive strategies--the production of subitaneously developing and resting eggs. Here, we hypothesize that both the somatic growth and the type of reproduction of the aquatic keystone herbivore Daphnia is limited by the availability of specific essential amino acids in the diet. In laboratory experiments, we investigated this hypothesis by feeding a high quality phytoplankton organism (Cryptomonas) and a green alga of moderate nutritional quality (Chlamydomonas) to a clone of Daphnia pulex with and without the addition of essential amino acids. The somatic growth of D. pulex differed between the algae of different nutritional quality, but not dependent on the addition of dissolved amino acids. However, in reproduction experiments, where moderate crowding conditions at saturating food quantities were applied, addition of the essential amino acids arginine and histidine (but not lysine and threonine) increased the total number and the developmental stage of subitaneous eggs. While D. pulex did not produce resting eggs on Cryptomonas, relatively high numbers of resting eggs were released on Chlamydomonas. When arginine and histidine were added to the green algal diet, the production of resting eggs was effectively suppressed. This demonstrates the high, but previously overlooked importance of single essential amino acids for the reproductive strategy of the aquatic keystone herbivore Daphnia. PMID:22163027

  7. 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

  8. 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

  9. Essential dietary amino acids for growth of larvae of the yellow mealworm, Tenebrio molitor L.

    PubMed

    Davis, G R

    1975-08-01

    Larvae of the yellow mealworm, Tenebrio molitor L., have been used to evaluate nutritional quality of proteins and protein isolates. However, such investigations have been complicated by lack of knowledge of dietary requirements of the larvae. To determine essential dietary amino acids for growth of Tenebrio molitor, single amino acids were deleted from the amino acid mixture of the diet. Diets were maintained isonitrogenous with supplementary glycine and, in the case of deleted glycine, with glutamic acid. Growth, as measured by gain in weight, and survival were observed over a 4-week period at 27 plus or minus 0.25 degrees and 65 plus or minus 5% relative humidity. The results indicate that larvae of Tenebrio molitor require a dietary source of the same 10 amino acids essential for growth in rats, other vertebrates, and some protozoa. They also showed that serine, tyrosine, glutamic acid, and possibly glycine were dispensable for growth in this insect. Alanine, cystine, proline, and aspartic acid appeared semidispensable. Survival over the 4-week experimental period was unaffected by deleting amino acids from the diet. The results are discussed in relation to amino acid requirements of other insects and to suggested improvement of the diet of the present investigation.

  10. Hepatic Fasting-Induced PPARα Activity Does Not Depend on Essential Fatty Acids.

    PubMed

    Polizzi, Arnaud; Fouché, Edwin; Ducheix, Simon; Lasserre, Frédéric; Marmugi, Alice P; Mselli-Lakhal, Laila; Loiseau, Nicolas; Wahli, Walter; Guillou, Hervé; Montagner, Alexandra

    2016-09-24

    The liver plays a central role in the regulation of fatty acid metabolism, which is highly sensitive to transcriptional responses to nutrients and hormones. Transcription factors involved in this process include nuclear hormone receptors. One such receptor, PPARα, which is highly expressed in the liver and activated by a variety of fatty acids, is a critical regulator of hepatic fatty acid catabolism during fasting. The present study compared the influence of dietary fatty acids and fasting on hepatic PPARα-dependent responses. Pparα(-/-) male mice and their wild-type controls were fed diets containing different fatty acids for 10 weeks prior to being subjected to fasting or normal feeding. In line with the role of PPARα in sensing dietary fatty acids, changes in chronic dietary fat consumption influenced liver damage during fasting. The changes were particularly marked in mice fed diets lacking essential fatty acids. However, fasting, rather than specific dietary fatty acids, induced acute PPARα activity in the liver. Taken together, the data imply that the potent signalling involved in triggering PPARα activity during fasting does not rely on essential fatty acid-derived ligand.

  11. Hepatic Fasting-Induced PPARα Activity Does Not Depend on Essential Fatty Acids

    PubMed Central

    Polizzi, Arnaud; Fouché, Edwin; Ducheix, Simon; Lasserre, Frédéric; Marmugi, Alice P.; Mselli-Lakhal, Laila; Loiseau, Nicolas; Wahli, Walter; Guillou, Hervé; Montagner, Alexandra

    2016-01-01

    The liver plays a central role in the regulation of fatty acid metabolism, which is highly sensitive to transcriptional responses to nutrients and hormones. Transcription factors involved in this process include nuclear hormone receptors. One such receptor, PPARα, which is highly expressed in the liver and activated by a variety of fatty acids, is a critical regulator of hepatic fatty acid catabolism during fasting. The present study compared the influence of dietary fatty acids and fasting on hepatic PPARα-dependent responses. Pparα−/− male mice and their wild-type controls were fed diets containing different fatty acids for 10 weeks prior to being subjected to fasting or normal feeding. In line with the role of PPARα in sensing dietary fatty acids, changes in chronic dietary fat consumption influenced liver damage during fasting. The changes were particularly marked in mice fed diets lacking essential fatty acids. However, fasting, rather than specific dietary fatty acids, induced acute PPARα activity in the liver. Taken together, the data imply that the potent signalling involved in triggering PPARα activity during fasting does not rely on essential fatty acid-derived ligand. PMID:27669233

  12. Hepatic Fasting-Induced PPARα Activity Does Not Depend on Essential Fatty Acids.

    PubMed

    Polizzi, Arnaud; Fouché, Edwin; Ducheix, Simon; Lasserre, Frédéric; Marmugi, Alice P; Mselli-Lakhal, Laila; Loiseau, Nicolas; Wahli, Walter; Guillou, Hervé; Montagner, Alexandra

    2016-01-01

    The liver plays a central role in the regulation of fatty acid metabolism, which is highly sensitive to transcriptional responses to nutrients and hormones. Transcription factors involved in this process include nuclear hormone receptors. One such receptor, PPARα, which is highly expressed in the liver and activated by a variety of fatty acids, is a critical regulator of hepatic fatty acid catabolism during fasting. The present study compared the influence of dietary fatty acids and fasting on hepatic PPARα-dependent responses. Pparα(-/-) male mice and their wild-type controls were fed diets containing different fatty acids for 10 weeks prior to being subjected to fasting or normal feeding. In line with the role of PPARα in sensing dietary fatty acids, changes in chronic dietary fat consumption influenced liver damage during fasting. The changes were particularly marked in mice fed diets lacking essential fatty acids. However, fasting, rather than specific dietary fatty acids, induced acute PPARα activity in the liver. Taken together, the data imply that the potent signalling involved in triggering PPARα activity during fasting does not rely on essential fatty acid-derived ligand. PMID:27669233

  13. 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

  14. 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

  15. 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

  16. Aging differentially affects human skeletal muscle amino acid transporter expression when essential amino acids are ingested after exercise

    PubMed Central

    Dickinson, Jared M.; Drummond, Micah J.; Coben, Jennifer R.; Volpi, Elena; Rasmussen, Blake B.

    2012-01-01

    Background & Aims Amino acid transporters have been proposed as regulators of protein synthesis. The primary aim of this study was to determine whether amino acid transporter expression is increased in human muscle following resistance exercise (RE) coupled with essential amino acid (EAA) ingestion, and whether a differential response occurs with aging. Secondly, we aimed to compare this response to a previous study examining RE alone. Methods Young (n=7, 30±2yr) and older men (n=6, 70±2yr) ingested EAA 1h after RE. Muscle biopsies were obtained at rest and 3 and 6h postexercise to examine amino acid transporter mRNA and protein expression. Results In both age groups, RE+EAA increased mRNA of L-type amino acid transporter 1 (LAT1)/solute linked carrier (SLC)7A5, sodium-coupled neutral amino acid transporter 2 (SNAT2)/SLC38A2, and cationic amino acid transporter 1/SLC7A1 (p<0.05). SNAT2 protein increased in young at 3 and 6h (p<0.05), whereas old maintained higher LAT1 protein (p<0.05). Compared to RE alone, RE+EAA enhanced amino acid transporter expression only in young (p<0.05). Conclusions RE increases muscle amino acid transporter expression in young and older adults, however, postexercise EAA ingestion enhances amino acid transporter expression only in young indicating that aging may influence the function of specific amino acid transporters. PMID:22889597

  17. Establishing an essential amino acid profile for maintenance in poultry using deletion method.

    PubMed

    Dorigam, J C P; Sakomura, N K; de Lima, M B; Sarcinelli, M F; Suzuki, R M

    2016-10-01

    This study aimed to estimate the essential amino acid profile and the ideal ratio for the maintenance of poultry by deletion method. A nitrogen balance (NB) trial was conducted using 198 adult roosters, housed individually in metabolic cages. The treatments were 33 purified diets being 11 diets with an amino acid mixture providing high protein intake of 500 mg N/BWkg (0.75) per day, 11 diets providing medium protein intake of 250 mg N/BWkg (0.75) per day (in each diet, one amino acid tested was diluted 50%) and 11 diets providing low protein intake of 125 mg N/BWkg (0.75) per day (made by omitting the amino acid tested). Each treatment had six replicates. After 48 h of fasting receiving water plus sucrose, the roosters were fed 40 g of the diets by tube once a day for 3 days. The excreta were collected within 72 h after the first feeding. The diets and excreta were analysed for nitrogen content. For each amino acid studied, a linear regression was fitted by NB and amino acid intake (AAI). The maintenance requirements were estimated as the AAI to maintain the NB equal to zero. The daily amino acid requirements for maintenance were estimated to be Lys 11, Met 29, Thr 23, Trp 5, Arg 50, Val 29, His 6, Gly 54, Phe 49, Leu 78 and Ile 21 mg/BWkg (0.75) per day. Therefore, the amino acid ratio for maintenance was concluded to be Lys 100, Met 276, Thr 220, Trp 48, Arg 467, Val 275, His 60, Gly 511, Phe 467, Leu 735 and Ile 198% independent of the scale. The essential amino acid profile and the ideal ratio for the maintenance of poultry estimated in this study contributed to improve the factorial model for estimating essential amino acid requirements for poultry.

  18. Enhancing essential amino acids and health benefit components in grain crops for improved nutritional values.

    PubMed

    Wenefrida, Ida; Utomo, Herry S; Blanche, Sterling B; Linscombe, S D

    2009-01-01

    Improving essential amino acids or protein content, along with other phytonutrients in the food crops, will affect a great portion of the world population, especially in developing countries where rice grain is the main source of protein. Malnutrition, including deficiencies in protein/energy, iron/zinc, vitamin A, and iodine, causes a total 24,000 deaths per day worldwide. The problem is severe where rice is the major staple food. Protein deficiency involves both the quantity (amount) and quality (the content in essential amino acids) of the dietary protein. Various interventions, such as distribution, fortification, dietary diversification, and measures against infectious diseases, have been applied to reduce deficiency disorders. The problem, however, remains unsolved. Developing genetically novel lines with elevated content of essential amino acids together with other health benefit components becomes more feasible for the enhancement of breeding techniques, genomics, molecular manipulations, and genetic engineering. Advancement in basic genetic and genetic engineering has resulted in successful enrichment of some essential amino acids, such as lysine (Lys), tryptophan (Trp), and methionine (Met). Successful genetic enhancement has been largely restricted to the maize crop through enrichment of grain Lys and to some extends Trp. Since rice is the main source of calories and protein intake for billions of people, enhancing essential amino acids in rice represents a tremendous challenge. This paper will discuss and review the current status in basic genetics, molecular genetics, and genetic engineering associated with the enhancement of amino acids and other health benefit components in major grain crop improvement. Patents and future efforts associated with enhancing nutritional quality of the grain will also be reviewed as a concerted effort to solve the malnutrition problem and improve the quality of life worldwide.

  19. 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.

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

    PubMed

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

    1994-08-01

    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.

  1. 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

  2. A microalgae residue based carbon solid acid catalyst for biodiesel production.

    PubMed

    Fu, Xiaobo; Li, Dianhong; Chen, Jie; Zhang, Yuanming; Huang, Weiya; Zhu, Yi; Yang, Jun; Zhang, Chengwu

    2013-10-01

    Biodiesel production from microalgae is recognized as one of the best solutions to deal with the energy crisis issues. However, after the oil extraction from the microalgae, the microalgae residue was generally discarded or burned. Here a novel carbon-based solid acid catalyst derived from microalgae residue by in situ hydrothermal partially carbonization were synthesized. The obtained catalyst was characterized and subjected to both the esterification of oleic acid and transesterification of triglyceride to produce biodiesel. The catalyst showed high catalytic activity and can be regenerated while its activity can be well maintained after five cycles.

  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. Dietary requirements of "nutritionally non-essential amino acids" by animals and humans.

    PubMed

    Wu, Guoyao; Wu, Zhenlong; Dai, Zhaolai; Yang, Ying; Wang, Weiwei; Liu, Chuang; Wang, Bin; Wang, Junjun; Yin, Yulong

    2013-04-01

    Amino acids are necessary for the survival, growth, development, reproduction and health of all organisms. They were traditionally classified as nutritionally essential or non-essential for mammals, birds and fish based on nitrogen balance or growth. It was assumed that all "non-essential amino acids (NEAA)" were synthesized sufficiently in the body to meet the needs for maximal growth and health. However, there has been no compelling experimental evidence to support this assumption over the past century. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and protect its mucosal integrity. Therefore, based on new research findings, NEAA should be taken into consideration in revising the classical "ideal protein" concept and formulating balanced diets to improve protein accretion, food efficiency, and health in animals and humans.

  5. Effects of essential fatty acid supplementation in dogs with idiopathic epilepsy: a clinical trial.

    PubMed

    Matthews, Helen; Granger, Nicolas; Wood, James; Skelly, Barbara

    2012-03-01

    The effects of essential fatty acid supplementation (EFA) on the control of idiopathic epilepsy in dogs were investigated in a blinded, placebo-controlled trial. Fifteen dogs were treated with triple purified Ω-3 oil containing 400 mg eicosapentaenoic acid, 250 mg docosahexaenoic acid and 22 mg vitamin E per 1.5 mL at a dose of 1.5 mL/10 kg once daily for 12 weeks, followed by a 12 week placebo period of supplementation with olive oil. Owners recorded seizure frequency and severity and any adverse events. EFA supplementation did not reduce seizure frequency or severity in dogs with idiopathic epilepsy.

  6. Triterpenoids, essential oil and photo-oxidative 28 --> 13-lactonization of oleanolic acid from Lantana camara.

    PubMed

    Misra, L; Laatsch, H

    2000-08-01

    Two novel triterpenoids have been isolated from the roots of Lantana camara L.: 3beta,19alpha dihydroxy ursan-28-oic acid and 21,22beta-epoxy-3beta-hydroxy olean-12-en-28-oic acid in its methyl ester form. Its leaves have yielded an essential oil which is rich in sesquiterpenes. Oleanolic acid, which is thought to be a hepatoprotective compound, was isolated from L. camara roots and converted into its 28 --> 13beta lactone by a facile photo-oxidation reaction.

  7. 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.

  8. Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues.

    PubMed

    Hughes, Austin L

    2014-07-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family.

  9. Differentiating N-terminal aspartic and isoaspartic acid residues in peptides.

    PubMed

    Sargaeva, Nadezda P; Lin, Cheng; O'Connor, Peter B

    2011-09-01

    Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(•) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+•) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+•).

  10. Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

    PubMed Central

    Hughes, Austin L.

    2015-01-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701

  11. 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

  12. Revised Backbone-Virtual-Bond-Angle Potentials to Treat the l- and d-Amino Acid Residues in the Coarse-Grained United Residue (UNRES) Force Field

    PubMed Central

    2015-01-01

    Continuing our effort to introduce d-amino-acid residues in the united residue (UNRES) force field developed in our laboratory, in this work the Cα ··· Cα ··· Cα backbone-virtual-bond-valence-angle (θ) potentials for systems containing d-amino-acid residues have been developed. The potentials were determined by integrating the combined energy surfaces of all possible triplets of terminally blocked glycine, alanine, and proline obtained with ab initio molecular quantum mechanics at the MP2/6-31G(d,p) level to calculate the corresponding potentials of mean force (PMFs). Subsequently, analytical expressions were fitted to the PMFs to give the virtual-bond-valence potentials to be used in UNRES. Alanine represented all types of amino-acid residues except glycine and proline. The blocking groups were either the N-acetyl and N′,N′-dimethyl or N-acetyl and pyrrolidyl group, depending on whether the residue next in sequence was an alanine-type or a proline residue. A total of 126 potentials (63 symmetry-unrelated potentials for each set of terminally blocking groups) were determined. Together with the torsional, double-torsional, and side-chain-rotamer potentials for polypeptide chains containing d-amino-acid residues determined in our earlier work (Sieradzan et al. J. Chem. Theory Comput., 2012, 8, 4746), the new virtual-bond-angle (θ) potentials now constitute the complete set of physics-based potentials with which to run coarse-grained simulations of systems containing d-amino-acid residues. The ability of the extended UNRES force field to reproduce thermodynamics of polypeptide systems with d-amino-acid residues was tested by comparing the experimentally measured and the calculated free energies of helix formation of model KLALKLALxxLKLALKLA peptides, where x denotes any d- or l- amino-acid residue. The obtained results demonstrate that the UNRES force field with the new potentials reproduce the changes of free energies of helix formation upon d

  13. 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

    2015-12-16

    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.

  14. 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

  15. Protective Effect of Ocimum basilicum Essential Oil Against Acetic Acid-Induced Colitis in Rats.

    PubMed

    Rashidian, Amir; Roohi, Parnia; Mehrzadi, Saeed; Ghannadi, Ali Reza; Minaiyan, Mohsen

    2016-10-01

    Ocimum basilicum L has been traditionally used for the treatment of inflammatory bowel disease in Iran. This study investigates the ameliorative effect of Ocimum basilicum essential oil on an acetic acid-induced colitis model in rats. Ocimum basilicum essential oil with 2 doses (200 and 400 μL/kg) significantly ameliorated wet weight/length ratio of colonic tissue compared to the control group. Higher doses of essential oil (200 and 400 μL/kg) significantly reduced ulcer severity, ulcer area, and ulcer index. On the other hand, histological examination revealed the diminution of total colitis index as a marker for inflammatory cell infiltration in the colonic segments of rats treated with Ocimum basilicum essential oil (200 and 400 μL/kg). The increased level of myeloperoxidase was significantly decreased after the treatment with the essential oil (200 and 400 μL/kg). These results suggest that Ocimum basilicum exhibits protective effect against acetic acid-induced colitis. PMID:26620574

  16. 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

  17. 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.)

  18. Function of aspartic acid residues in optimum pH control of L-arabinose isomerase from Lactobacillus fermentum.

    PubMed

    Xu, Zheng; Li, Sha; Feng, Xiaohai; Zhan, Yijing; Xu, Hong

    2014-05-01

    L-Arabinose isomerase (L-AI) catalyzes the isomerization of L-arabinose to L-ribulose and D-galactose to D-tagatose. Most reported L-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial D-tagatose production. Lactobacillus fermentum L-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for D-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, D-galactose was isomerized into D-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other L-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of L-AIs that can be modified for desired optimum pH and better pH stability, which are useful in D-tagatose bioproduction.

  19. Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.

    PubMed

    Nichols, K; Kim, J J M; Carson, M; Metcalf, J A; Cant, J P; Doelman, J

    2016-02-01

    To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

  20. Decrease in essential fatty acid content of edible fats during the frying process.

    PubMed

    Gere, A

    1982-09-01

    Degradation of sunflower oil, rapeseed oil, and lard during the frying operation was investigated by studying the loss of essential fatty acids compared to the accumulation of decomposition products. Linoleic and/or linolenic acid concentration was measured by GLC, and for detecting decomposition products determination of polymer content by GPC was chosen. Twelve laboratory experiments with different heating or frying conditions were run aimed at modelling practice and studying the effects of certain factors. The results indicated that loss of essential fatty acids being a parallel process to the accumulation of breakdown products is suitable both for detecting the decrease in nutritive value and for quality assessment of used frying fats. It was also found that the rate of deterioration is considerably affected by the nature of fat and the frying parameters. Study of the relationship between polymer content and the decrease in essential fatty acid concentration (using data from 110 samples) showed that high, linear correlation depending on the nature of fat can be found. Correlation coefficients and equations of regression lines were calculated.

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  2. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  3. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  4. Polyunsaturated fatty acids in human milk: an essential role in infant development.

    PubMed

    Innis, Sheila M

    2004-01-01

    The n-6 and n-3 fatty acids are essential dietary nutrients required for optimal growth and development, particularly of the brain and retina. Large amounts of the n-3 fatty acid docosahexaenoic acid (DHA) is accumulated in the brain grey matter and the visual elements of the retina during development, and reduced DHA in these tissues can result in decreased visual and psychomotor development. Although the possible importance of differences in n-6 and n-3 fatty acids, particularly DHA, between human milk and infant formulas has been the subject of intense clinical research, the variability in the essential fatty acid content of milk within and among different populations of women and implications of this to infant growth and development have received much less attention. Considerable research has shown that the DHA content of the maternal diet is the most important determinant of the amount of DHA secreted in milk, and thus the dietary intake of the breastfed infant. The DHA content of human milk varies over 10-fold, being lowest in women with no intake of DHA and highest in women with high intakes of DHA, which is found predominantly in fatty fish. The requirement for n-3 fatty acids, and the balance of n-6 and n-3 fatty acids for optimal growth and development of the brain and retina, and long-term minimization of risk of chronic disease remains as one of the most important questions in infant nutrition. Dietary recommendations to modifying dietary fat with the aim of reducing risk of chronic disease, including obesity and cardiovascular disease in adults, need to consider that when followed by pregnant women, these recommendations can have a marked effect on the amount and balance of n-6 and n-3 fatty acids secreted in milk.

  5. 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

  6. 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

  7. FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function.

    PubMed

    Banerjee, Antara A; Dupakuntla, Madhavi; Pathak, Bhakti R; Mahale, Smita D

    2015-06-01

    The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH-FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L(501)F mainly and I(505)V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L(501)F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function. PMID:25791375

  8. Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

    PubMed

    To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

    2011-05-01

    Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

  9. 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

  10. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

    PubMed Central

    Engelman, A; Craigie, R

    1992-01-01

    We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration. Images PMID:1404595

  11. 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

  12. 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

  13. 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.

  14. Essential fatty acid deficiency while a patient receiving fat regimen total parenteral nutrition

    PubMed Central

    Roongpisuthipong, Wanjarus; Phanachet, Pariya; Roongpisuthipong, Chulaporn; Rajatanavin, Natta

    2012-01-01

    A 32-year-old man was diagnosed with lymphoma and underwent Billroth’s II operation because of upper gastrointestinal haemorrhage. Although the patient received fat regimen total parenteral nutrition (TPN), the patient developed typical skin rash of essential fatty acid deficiency after 2 weeks of starting TPN. The diagnosis was confirmed by biochemical and histological analyses. After increasing the lipid infusion, the rash was gradually improved with complete resolution after 19 days. PMID:22707694

  15. Physicochemical pretreatments and hydrolysis of furfural residues via carbon-based sulfonated solid acid.

    PubMed

    Ma, Bao Jun; Sun, Yuan; Lin, Ke Ying; Li, Bing; Liu, Wan Yi

    2014-03-01

    Potential commercial physicochemical pretreatment methods, NaOH/microwave and NaOH/ultrasound were developed, and the carbon-based sulfonated solid acid catalysts were prepared for furfural residues conversion into reducing sugars. After the two optimum pretreatments, both the content of cellulose increased (74.03%, 72.28%, respectively) and the content of hemicellulose (94.11%, 94.17% of removal rate, respectively) and lignin (91.75%, 92.09% of removal rate, respectively) decreased in furfural residues. The reducing sugar yields of furfural residues with the two physicochemical pretreatments on coal tar-based solid acid reached 33.94% and 33.13%, respectively, higher than that pretreated via NaOH alone (27%) and comparable to that pretreated via NaOH/H2O2 (35.67%). The XRD patterns, IR spectra and SEM images show microwave and ultrasound improve the pretreatment effect. The results demonstrate the carbon-based sulfonated solid acids and the physicochemical pretreatments are green, effective, low-cost for furfural residues conversion.

  16. Mitochondrial lipoic acid scavenging is essential for Plasmodium berghei liver stage development.

    PubMed

    Deschermeier, Christina; Hecht, Leonie-Sophie; Bach, Frauke; Rützel, Katharina; Stanway, Rebecca R; Nagel, Andreas; Seeber, Frank; Heussler, Volker T

    2012-03-01

    Lipoic acid is an essential cofactor for enzymes that participate in key metabolic pathways in most organisms. While in mammalian cells lipoylated proteins reside exclusively in the mitochondria, apicomplexan parasites of the genus Plasmodium harbour two independent lipoylation pathways in the mitochondrion and the apicoplast, a second organelle of endosymbiotic origin. Protein lipoylation in the apicoplast relies on de novo lipoic acid synthesis while lipoylation of proteins in the mitochondrion depends on scavenging of lipoic acid from the host cell. Here, we analyse the impact of lipoic acid scavenging on the development of Plasmodium berghei liver stage parasites. Treatment of P. berghei-infected HepG2 cells with the lipoic acid analogue 8-bromo-octanoic acid (8-BOA) abolished lipoylation of mitochondrial enzyme complexes in the parasite while lipoylation of apicoplast proteins was not affected. Parasite growth as well as the ability of the parasites to successfully complete liver stage development by merosome formation were severely impaired but not completely blocked by 8-BOA. Liver stage parasites were most sensitive to 8-BOA treatment during schizogony, the phase of development when the parasite grows and undergoes extensive nuclear division to form a multinucleated syncytium. Live cell imaging as well as immunofluorescence analysis and electronmicroscopy studies revealed a close association of both host cell and parasite mitochondria with the parasitophorous vacuole membrane suggesting that host cell mitochondria might be involved in lipoic acid uptake by the parasite from the host cell.

  17. Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B.

    PubMed

    Sikorra, Stefan; Henke, Tina; Swaminathan, Subramanyam; Galli, Thierry; Binz, Thomas

    2006-03-24

    Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.

  18. [The intake of proteins and essential amino acids in top-competing women athletes].

    PubMed

    Nuviala Mateo, R J; Lapieza Laínez, M G

    1997-01-01

    In a group of 84 sportswomen (19 karatekas, 20 handball and 20 basketball players and 25 middle- and long-distance runners) the intake of protein and essential amino acids was assessed, as well as the urinary excretion of urea and creatinine. Simultaneously, we have studied 82 women who did not underwent physical activity (control group). After a 7-days long recall, by means of the food weighing method, was assessed by a computerized program the daily average intake of: total energy, macronutrients, proteins of animal origin and essential amino acids. The determination of urinary urea and creatinine was made in a 24-hours sample. The protein intake was significantly higher (p < 0.01) in basketball and handball players and in the runners than in the control group. The protein intake related with the body weight (g/kg) was significantly higher in basketball players (p < 0.05) and in the runners (p < 0.01), taking both the sportswomen and the sedentary women quantities higher than 0.8 g/kg/day established as minimum allowances. The amino acids intake related with the body weight (mg/kg), exceeded widely in all the studied groups the minimum recommended for each of them, outstanding the values significantly high (p < 0,01) found for all the amino acids in the runners. Both for the urinary excretion of the urea and the creatinine, the highest values correspond to the runners. All the sportswomen have superior protein and essential amino acids intake in relation with women without physical activity. The higher body losses of nitrogen in the form of urea and creatinine found in the runners are due to their higher protein/kg of body weight intake.

  19. Essential fatty acid requirements of vegetarians in pregnancy, lactation, and infancy.

    PubMed

    Sanders, T A

    1999-09-01

    Long-chain polyunsaturated fatty acids (LCPUFAs) derived from linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids are required for the normal development of the retina and central nervous system, but the extent to which they can be synthesized from the parent fatty acids is debated. Consuming LCPUFAs markedly increases their proportions in tissue lipids compared with their parent fatty acids. Thus, it has been argued that LCPUFAs must be supplied in the diet. LCPUFAs are generally absent from plant foods, thus it is important find out how essential fatty acid requirements are met by vegetarians. A developing fetus obtains LCPUFAs via selective uptake from its mother's plasma and LCPUFAs are present in the breast milk of vegetarians. There is no evidence that the capacity to synthesize LCPUFAs is limited in vegetarians. However, there are greater proportions of n-6 LCPUFAs and lower proportions of n-3 LCPUFAs in vegetarians compared with omnivores. This difference is probably a consequence of the selection of foods by vegetarians with high amounts of linoleic acid. Although lower concentrations of docosahexaenoic acid (22:6n-3; DHA) have been observed in blood and artery phospholipids of infants of vegetarians, it is uncertain whether their brain lipids contain lower proportions of DHA than do those of infants of omnivores. On the basis of experiments in primates that showed altered visual function with a high ratio of linoleic acid to alpha-linolenic acid, it would be prudent to recommend diets with a ratio between 4:1 and 10:1 in vegetarians and that excessive intakes of linoleic acid be avoided.

  20. 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

  1. The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

    PubMed

    Brosh, R M; Balajee, A S; Selzer, R R; Sunesen, M; Proietti De Santis, L; Bohr, V A

    1999-11-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  2. Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue.

    PubMed

    Pasta, P; Mazzola, G; Carrea, G

    1987-03-10

    Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

    PubMed

    Somajo, Sofia; Ahnström, Josefin; Fernandez-Recio, Juan; Gierula, Magdalena; Villoutreix, Bruno O; Dahlbäck, Björn

    2015-05-01

    Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

  4. Oxygen consumption constrains food intake in fish fed diets varying in essential amino acid composition.

    PubMed

    Saravanan, Subramanian; Geurden, Inge; Figueiredo-Silva, A Cláudia; Nusantoro, Suluh; Kaushik, Sadasivam; Verreth, Johan; Schrama, Johan W

    2013-01-01

    Compromisation of food intake when confronted with diets deficient in essential amino acids is a common response of fish and other animals, but the underlying physiological factors are poorly understood. We hypothesize that oxygen consumption of fish is a possible physiological factor constraining food intake. To verify, we assessed the food intake and oxygen consumption of rainbow trout fed to satiation with diets which differed in essential amino acid (methionine and lysine) compositions: a balanced vs. an imbalanced amino acid diet. Both diets were tested at two water oxygen levels: hypoxia vs. normoxia. Trout consumed 29% less food under hypoxia compared to normoxia (p<0.001). Under both hypoxia and normoxia trout significantly reduced food intake by 11% and 16% respectively when fed the imbalanced compared to the balanced amino acid diet. Oxygen consumption of the trout per unit body mass remained identical for both diet groups not only under hypoxia but also under normoxia (p>0.05). This difference in food intake between diets under normoxia together with the identical oxygen consumption supports the hypothesis that food intake in fish can be constrained by a set-point value of oxygen consumption, as seen here on a six-week time scale.

  5. 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

  6. Autistic Children Exhibit Decreased Levels of Essential Fatty Acids in Red Blood Cells

    PubMed Central

    Brigandi, Sarah A.; Shao, Hong; Qian, Steven Y.; Shen, Yiping; Wu, Bai-Lin; Kang, Jing X.

    2015-01-01

    Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3–17. Analysis of the fatty acid composition of red blood cell (RBC) membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA) were particularly decreased (p < 0.001). In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2) were higher in a subset of the autistic participants (n = 20) compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism. PMID:25946342

  7. Effect of dietary protected lipids on the essential fatty acid status of the newborn kid.

    PubMed

    Soares, M C

    1986-08-01

    Pregnant goats were fed either a control diet or one in which part of the concentrate ration was replaced, for either the last 2 mo or the last month of gestation, with a polyunsaturated fatty acid supplement protected from biohydrogenation in the rumen. On the day of parturition, goats fed a supplemented diet, during either 1 or 2 mo before parturition, exhibited markedly higher concentrations of linoleic acid in the major plasma lipid fractions, i.e., phospholipids, cholesteryl esters, triacylglycerols and nonesterified fatty acids, than did goats not fed the supplement. At birth, the four main lipid fractions in the plasma of the kids from goats fed the supplemented diet, during either 1 or 2 mo before parturition, exhibited considerably higher proportions of linoleic and arachidonic acids and lower proportions of eicosatrienoic acid than did those of newborn kids from unsupplemented mothers. These findings show appreciable transfer of lipids across the placenta and demonstrate that the low essential fatty acids status of kids at birth can be raised by this particular manipulation of the maternal diet.

  8. Essential fatty acid profiles differ across diets and browse of black rhinoceros.

    PubMed

    Grant, Jacqualine B; Brown, Dan L; Dierenfeld, Ellen S

    2002-01-01

    In captivity, black rhinoceros (Diceros bicornis) suffer from idiopathic skin lesions that may be linked to dietary deficiencies, in particular essential fatty acid deficiency (EFAD). Therefore, a study was undertaken from July 1995 to May 1997 to characterize the diet of captive D. bicornis in North American zoos and measure fat and fatty acid composition in zoo diet, and African and North American browses. Descriptions of all dietary items offered to black rhinos on a daily basis were compiled from 20 North American zoos; zoo diet contained (mean +/- SE) 61 +/- 2% hay, 28 +/- 2% grain pellets, 6 +/- 1% produce, and 5 +/- 1% fresh browse, with hay and grain pellets together comprising nearly 90% of items offered. Gas chromatography-mass spectrometry analysis (GC-MS) was used to measure triacylglycerol equivalent (TAG), total fatty acids (TFA), and essential fatty acids (EFA) in zoo diet, and African and North American browses. North American browse contained more TAG and TFA than did zoo diet or African browse. Zoo diet contained more linoleic acid (18:2n6) and less linolenic acid (18:3n3) than either African browse corrected for degradation losses or North American browse, whether measured as weight percentage of dry sample or as weight percentage of TFA. In addition, the ratio of 18:2n6 to 18:3n3 was significantly lower in both browses than in zoo diet. There are significant nutritional differences between the major dietary components of North American captive black rhinoceros diets and native African browses that warrant further exploration given the health problems associated with this animal in captivity. PMID:11838204

  9. Essential fatty acid profiles differ across diets and browse of black rhinoceros.

    PubMed

    Grant, Jacqualine B; Brown, Dan L; Dierenfeld, Ellen S

    2002-01-01

    In captivity, black rhinoceros (Diceros bicornis) suffer from idiopathic skin lesions that may be linked to dietary deficiencies, in particular essential fatty acid deficiency (EFAD). Therefore, a study was undertaken from July 1995 to May 1997 to characterize the diet of captive D. bicornis in North American zoos and measure fat and fatty acid composition in zoo diet, and African and North American browses. Descriptions of all dietary items offered to black rhinos on a daily basis were compiled from 20 North American zoos; zoo diet contained (mean +/- SE) 61 +/- 2% hay, 28 +/- 2% grain pellets, 6 +/- 1% produce, and 5 +/- 1% fresh browse, with hay and grain pellets together comprising nearly 90% of items offered. Gas chromatography-mass spectrometry analysis (GC-MS) was used to measure triacylglycerol equivalent (TAG), total fatty acids (TFA), and essential fatty acids (EFA) in zoo diet, and African and North American browses. North American browse contained more TAG and TFA than did zoo diet or African browse. Zoo diet contained more linoleic acid (18:2n6) and less linolenic acid (18:3n3) than either African browse corrected for degradation losses or North American browse, whether measured as weight percentage of dry sample or as weight percentage of TFA. In addition, the ratio of 18:2n6 to 18:3n3 was significantly lower in both browses than in zoo diet. There are significant nutritional differences between the major dietary components of North American captive black rhinoceros diets and native African browses that warrant further exploration given the health problems associated with this animal in captivity.

  10. 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.

  11. Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.

    PubMed

    Wang, Hou-yu; Li, Si; Tang, Yun-yun; Dong, Jing-yu; Fan, Liu-yin; Cao, Cheng-xi

    2013-06-21

    As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.

  12. Comparison between liquid and solid acids catalysts on reducing sugars conversion from furfural residues via pretreatments.

    PubMed

    Lin, Keying; Ma, Baojun; Sun, Yuan; Liu, Wanyi

    2014-09-01

    Liquid sulphuric acid is adopted and compared with carbon-based sulfonated solid acids (coal tar-based and active carbon-based) for furfural residues conversion into reducing sugars. The optimum hydrolysis conditions of liquid acid are at 4% of sulphuric acid, 25:1 of liquid and solid ratio, 175°C of reaction temperature and 120 min of reaction time. The reducing sugar yields are reached over 60% on liquid acid via NaOH/H2O2, NaOH/microwave and NaOH/ultrasonic pretreatments, whereas only over 30% on solid acids. The TOFs (turnover number frequency) via NaOH/H2O2 pretreatments are 0.093, 0.020 and 0.023 h(-1) for liquid sulphuric acid, coal tar-based and active carbon-based solid acids catalysts, respectively. Considering the efficiency, cost and environment factors, the liquid and solid acids have their own advantages of potential commercial application values.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Ketogenic Essential Amino Acids Modulate Lipid Synthetic Pathways and Prevent Hepatic Steatosis in Mice

    PubMed Central

    Kimura, Yoshiko; Aleman, Jose O.; Young, Jamey D.; Koyama, Naoto; Kelleher, Joanne K.; Takahashi, Michio; Stephanopoulos, Gregory

    2010-01-01

    Background Although dietary ketogenic essential amino acid (KAA) content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated. Methodology/Principal Findings We designed a diet with a high ratio (E/N) of essential amino acids (EAAs) to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr) without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogensis (DNL) fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides. Conclusion Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways. PMID:20706589

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Removal of copper from acid wastewater of bioleaching by adsorption onto ramie residue and uptake by Trichoderma viride.

    PubMed

    Wang, Buyun; Wang, Kai

    2013-05-01

    A continuous batch bioleaching was built to realize the bioleaching of sewage sludge in large scale. In the treatment, heavy metal in acid wastewater of bioleaching was removed by adsorption onto ramie residue. Then, acid wastewater was reused in next bioleaching batch. In this way, most time and water of bioleaching was saved and leaching efficiency of copper, lead and chromium kept at a high level in continuous batch bioleaching. It was found that residual heavy metal in sewage sludge is highly related to that in acid wastewater after bioleaching. To get a high leaching efficiency, concentration of heavy metal in acid wastewater should be low. Adsorption of copper from acid wastewater onto ramie residue can be described by pseudo first-order kinetics equation and Freundlich isotherm model. Trichoderma viride has the potential to be used for the concentration and recovery of heavy metal adsorbed onto ramie residue. PMID:23567687

  5. Extension of UNRES force field to treat polypeptide chains with D-amino-acid residues

    PubMed Central

    Sieradzan, Adam K.; Hansmann, Ulrich H.E.; Scheraga, Harold A.; Liwo, Adam

    2013-01-01

    Coarse-grained force fields for protein simulations are usually designed and parameterized to treat proteins composed of natural L-amino-acid residues. However, D-amino-acid residues occur in bacterial, fungal (e.g., gramicidins), as well as human-designed proteins. For this reason, we have extended the UNRES coarse-grained force field developed in our laboratory to treat systems with D-amino-acid residues. We developed the respective virtual-bond-torsional and double-torsional potentials for rotation about the Cα · · · Cα virtual-bond axis and two consecutive Cα · · · Cα virtual-bond axes, respectively, as functions of virtual-bond-dihedral angles γ. In turn, these were calculated as potentials of mean force (PMFs) from the diabatic energy surfaces of terminally-blocked model compounds for glycine, alanine, and proline. The potential-energy surfaces were calculated by using the ab initio method of molecular quantum mechanics at the Møller-Plesset (MP2) level of theory and the 6-31G(d,p) basis set, with the rotation angles of the peptide groups about Ci-1α⋯Ciα(λ(1)) and Ciα⋯Ci+1α(λ(2)) used as variables, and the energy was minimized with respect to the remaining degrees of freedom. The PMFs were calculated by numerical integration for all pairs and triplets with all possible combinations of types (glycine, alanine, and proline) and chirality (D or L); however, symmetry relations reduce the number of non-equivalent torsional potentials to 13 and the number of double-torsional potentials to 63 for a given C-terminal blocking group. Subsequently, one- (for torsional) and two-dimensional (for double-torsional potentials) Fourier series were fitted to the PMFs to obtain analytical expressions. It was found that the torsional potentials of the x-Y and X-y types, where X and Y are Ala or Pro, respectively, and a lowercase letter denotes D-chirality, have global minima for small absolute values of γ, accounting for the double-helical structure of

  6. Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighbouring the acceptor GalNAc residues

    PubMed Central

    2004-01-01

    C4ST-1 (chondroitin 4-sulphotransferase-1) and C6ST-1 (chondroitin 6-sulphotransferase-1) transfer sulphate from PAPS (adenosine 3′-phosphate 5′-phosphosulphate) to positions 4 and 6 respectively of the GalNAc residues of chondroitin. We showed previously that C4ST-1 purified from rat chondrosarcoma and recombinant C4ST-1 both transfer sulphate efficiently to position 4 of the GalNAc residues of DSDS (desulphated dermatan sulphate). We report here the specificity of C4ST-1 and C6ST-1 in terms of uronic acid residue recognition around the GalNAc residue to which sulphate is transferred. When [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C4ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in disaccharide fractions and the remainder distributed to tetrasaccharides and larger fractions, indicating that C4ST-1 mainly transferred sulphate to position 4 of the GalNAc residue located at the GlcA-GalNAc-GlcA sequence. Structural analysis of tetrasaccharide and larger oligosaccharide fractions indicated that C4ST-1 mainly transferred sulphate to the GalNAc residue adjacent to the reducing side of the GlcA residue. On the other hand, when [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C6ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in fractions larger than hexasaccharides, indicating that C6ST-1 transferred sulphate to the GalNAc residues located in the L-iduronic acid-rich region. Structural analysis of the tetrasaccharide and larger oligosaccharide fractions indicated that C6ST-1 showed very little preference for the GalNAc residue neighbouring the GlcA residue. These results indicate that C4ST-1 and C6ST-1 differ from each other in the recognition of uronic acid residues adjacent to the targeted GalNAc residue. PMID:15324304

  7. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  8. Essential fatty acid deficiency delays the onset of puberty in the female rat.

    PubMed

    Smith, S S; Neuringer, M; Ojeda, S R

    1989-09-01

    This study assessed the effect of a dietary deficiency in the essential fatty acids (EFA) linoleic and linolenic acids on the onset of female puberty. EFA deficiency was produced in female rats by means of a semipurified diet and was biochemically documented by analyzing serum and erythrocyte fatty acid levels of more than 30 fatty acids, including all members of the n-6 and n-3 series. Levels of linoleic acid (18:2 n-6) and all n-6 derivatives, particularly arachidonic acid, were strikingly reduced. A less pronounced but clear-cut decrease in n-3 fatty acids, including docosahexaenoic acid (22:6 n-3) was also found. The times of puberty and first ovulation, as assessed by the ages at vaginal opening and first diestrus, were significantly delayed in EFA-deficient rats. The mechanisms underlying this delay appear to reside at both hypothalamic and ovarian sites. Simulation of preovulatory plasma estradiol (E2) levels via implantation of E2-containing Silastic capsules evoked a LH surge 30 h later in control juvenile rats, but not in EFA-deficient animals, indicating a delay in the development of the hypothalamic component of E2-positive feedback in the latter group. This delay appears to be due at least in part to reduced prostaglandin E2 (PGE2) synthesis, as the ability of the neurotransmitter norepinephrine to induce PGE2 release from median eminence nerve terminals was markedly reduced in EFA-deficient rats compared with that in controls. The decrease in hypothalamic PGE2 release was related to the EFA deficiency and not to reduced PG synthase activity, as determined by HPLC analysis of PG synthase products derived from exogenous [14C]arachidonic acid. Basal and hCG-stimulated PGE2 synthesis was also compromised in ovaries from EFA-deficient rats. Depressed gonadal function resulting from the EFA deficiency was further evidenced by a reduced gonadotropin receptor content, a blunted E2 response to hCG in vitro, and an increase in mean serum FSH levels. These

  9. 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

  10. 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.

  11. Study of TATP: method for determination of residual acids in TATP.

    PubMed

    Matyáš, Robert; Chýlková, Jaromíra

    2013-05-10

    Triacetone triperoxide (3,3,6,6,9,9-hexamethyl-1,2,4,5,7,8-hexoxonane, TATP) is nowadays one of the most commonly used improvised explosives. It is prepared by the action of hydrogen peroxide on acetone in an acidic environment. Easily available mineral acids - hydrochloric, sulfuric, nitric and perchloric - are the most often recommended on the extremist web pages dealing with improvised production of explosives. The various TATP producers' choice of acid mainly depends on the author's experiences and the local availability of the acid. A knowledge of the kind of acid used for TATP production can help in detecting the person who has made the TATP, or who has committed a criminal act using TATP. Therefore, a capillary isotachophoretic method was developed for determination of residual anions (originating from the acid used during TATP synthesis) in the resulting TATP crystals. This analytical method has proved to be reliable; the acid used for TATP synthesis was correctly identified in all samples analyzed. PMID:23542054

  12. Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

    PubMed Central

    Hamada, Kenji; Terashima, Hiromichi; Arisawa, Mikio; Yabuki, Nami; Kitada, Kunio

    1999-01-01

    The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization. PMID:10383953

  13. Essential role of amino acid position 226 in oligosaccharide elongation by amylosucrase from Neisseria polysaccharea.

    PubMed

    Cambon, Emmanuelle; Barbe, Sophie; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; André, Isabelle

    2014-09-01

    Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose-like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single-mutants had a higher activity on sucrose compared to the wild-type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild-type enzyme, the R226N mutant showed a 10-fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose-like polymer that can be of interest for biotechnological applications.

  14. Effect of daily food supplementation with essential fatty acids on canine semen quality.

    PubMed

    da Rocha, A A; da Cunha, I C N; Ederli, B B; Albernaz, A P; Quirino, C R

    2009-07-01

    Polyunsaturated fatty acids are important membrane components that influence membrane integrity and fluidity. In the present study, the effect of oral supplementation for 60 days with essential fatty acids (omega 3, 6 and 9) and vitamin E on canine semen quality was evaluated. Sixteen dogs were selected for the experiment; eight were used as the control group and eight received the fatty acid supplemented diet for 60 days. Semen samples were taken every 15 days during the entire experimental period and were analyzed for volume (ml), motility (%), vigour (0-5), concentration (x10(6)/ml), morphology of spermatozoa (%), plasma membrane integrity (%; using the hyposmotic swelling test) and thermoresistance (motility and vigour after 4 h at 38 degrees C). We concluded that, daily supplementation with omega 3, omega 6 and omega 9 fatty acids, together with vitamin E, for a period of 60 days, significantly increased the semen volume of the treated group after 15 days of supplementation; the vigour and concentration of spermatozoa were superior after the first month of supplementation, while the percentage of morphologically abnormal spermatozoa decreased and the cells were protected against thermal stress.

  15. Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

    PubMed

    Allam, Uday Sankar; Krishna, M Gopala; Sen, Minakshi; Thomas, Rony; Lahiri, Amit; Gnanadhas, Divya Prakash; Chakravortty, Dipshikha

    2012-01-01

    During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the ΔSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The ΔSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the ΔSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the ΔSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

  16. Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

    PubMed

    Nguyen, Cuong Mai; Nguyen, Thanh Ngoc; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Park, Youn-Je; Kim, Jin-Cheol

    2014-01-01

    This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

  17. Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

    PubMed

    Nguyen, Cuong Mai; Nguyen, Thanh Ngoc; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Park, Youn-Je; Kim, Jin-Cheol

    2014-01-01

    This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter. PMID:24240182

  18. 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.

  19. 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.

  20. [Determination of clavulanic acid residue in milk by high performance liquid chromatography-tandem mass spectrometry].

    PubMed

    Yang, Gang; Huang, Xianhui; Guo, Chunna; Fang, Qiuhua; He, Limin

    2012-06-01

    An analytical method was developed for the determination of clavulanic acid (CLAV) in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A 2 g milk sample was deproteinized by ethanol. The supernatant was transferred into a pear-shaped bottle to be evaporated to about 0.5 mL, and the residue was dissolved with ammonium acetate solution. The sample was determined by HPLC-MS/MS after the purification. The chromatographic separation was achieved on a Luna 5u C8 column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution. The identification of CLAV was carried out by MS/MS equipped with electrospray ionization in negative scanning and multiple reaction monitoring (MRM) modes. Matrix-matched calibration standard was used for the quantification. The calibration curve showed perfect linear in the range of 10 - 400 microg/kg with the correlation coefficient of 0.999. The limit of detection (LOD, S/N > or = 3) was 10 microg/kg in milk, and the limit of quantification (LOQ, S/N > or = 10) was 20 microg/kg. The mean recoveries varied from 80.00% to 91.25% at the four spiked levels of LOQ, 1/2MRL (the maximum residue limit), MRL, and 2MRL with the relative standard deviations of 5.60% -8.77%. In conclusion, the established method can be applied for the determination of CLAV residues in milk.

  1. 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.

  2. 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.

  3. [Nitrate nitrogen leaching and residue of humic acid fertilizer in field soil].

    PubMed

    Liu, Fang-chun; Xing, Shang-jun; Duan, Chun-hua; Du, Zhen-yu; Ma, Hai-lin; Ma, Bing-yao

    2010-07-01

    To elucidate the potential influence of humic acidfertilizer on groundwater and soil quality in clay soil (CS) and sandy soil (SS), nitrate nitrogen leaching and residue of different fertilizers in field soil were studied using a self-made leaching field device. Nitrate nitrogen concentration in leaching water of fertilizer treatments was 28.1%-222.2% higher than that of non-nitrogen treatment in different times, but humic acid fertilizer could prevent nitrate nitrogen leaching both in CS and SS, especially in CS. Nitrate nitrogen concentration of leaching water in CS was 41.2%-59.1% less than that in SS and the inhibiting effect in CS was greater than that in SS. Nitrate nitrogen could be accumulated in soil profile by fertilizer application. The residue of nitrate nitrogen retained in 0-40 cm soil layer of humic acid fertilizer treatment was 59.8% and 54.4% respectively, higher than that of urea and compound fertilizer treatments. Nitrate nitrogen amount of humic acid, urea and compound fertilizer treatments in SS was significantly less than that in CS, being 81.7%, 81.1% and 47.6% respectively. Compared with the conventional fertilizer, humic acid fertilizer treatment improved the contents of organic matter, available nitrogen, phosphorus, and potassium of upper layer soil as well as cation exchange capacity. Besides, total amount of water-soluble salts in humic acid fertilizer treatment was decreased by 24.8% and 22.5% in comparison to urea and compound fertilizer treatments in CS, respectively. In summary, the application of humic acid fertilizer could improve physical and chemical properties of upper layer soil and reduce the risk of potential pollution to groundwater.

  4. A clamp-like orientation of basic residues set in a parallelogram is essential for heparin binding.

    PubMed

    Cheng, Yi-Yun; Cheng, Chao-Sheng; Lee, Tian-Ren; Chang, Wun-Shaing Wayne; Lyu, Ping-Chiang

    2016-09-01

    While the majority of studies have focused on the biological roles of heparin-binding proteins, relatively little is known about their key residues and structural elements responsible for heparin interaction. In this study, we employed the IgG-binding domain B1 of Streptococcal protein G as a miniature scaffold to investigate how certain positively charged residues within the β-sheet conformation become favorable for heparin binding. By performing a series of arginine substitution mutations followed by gain-of-heparin-binding analysis, we deduced that a clamp-like orientation with discontinuous basic residues separated by ~ 5 Å with ~ 100° interior angle is advantageous for high heparin affinity. PMID:27531580

  5. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism

    PubMed Central

    Stäubert, Claudia; Broom, Oliver Jay; Nordström, Anders

    2015-01-01

    Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them. We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation. We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death. Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy. PMID:25839160

  6. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism.

    PubMed

    Stäubert, Claudia; Broom, Oliver Jay; Nordström, Anders

    2015-08-14

    Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.

  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. 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

  9. Type II fatty acid synthesis is essential for the replication of Chlamydia trachomatis.

    PubMed

    Yao, Jiangwei; Abdelrahman, Yasser M; Robertson, Rosanna M; Cox, John V; Belland, Robert J; White, Stephen W; Rock, Charles O

    2014-08-01

    The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.

  10. 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.

  11. The Effect of varying ratios of docosahexaenoic Acid and arachidonic acid in the prevention and reversal of biochemical essential fatty acid deficiency in a murine model

    PubMed Central

    Le, Hau D.; Fallon, Erica M.; Kalish, Brian T.; de Meijer, Vincent E.; Meisel, Jonathan A.; Gura, Kathleen M.; Nose, Vania; Pan, Amy H.; Bistrian, Bruce R.; Puder, Mark

    2012-01-01

    Objective Essential fatty acids (EFA) are necessary for growth, development, and biological function, and must be acquired through the diet. While linoleic acid (LA) and alpha-linolenic acid (ALA) have been considered the true EFAs, we previously demonstrated that docosahexaenoic acid (DHA) and arachidonic acid (AA) taken together as the sole source of dietary fatty acids can prevent biochemical essential fatty acid deficiency (EFAD). This study evaluates the effect of varying dietary ratios of DHA:AA in the prevention and reversal of biochemical EFAD in a murine model. Methods Using a murine model of EFAD, we provided mice with 2.1% of daily caloric intake in varying DHA:AA ratios (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) for 19 days in association with a liquid high-carbohydrate fat-free diet to evaluate the effect on fatty acid profiles. In a second experiment, we evaluated the provision of varying DHA:AA ratios (20:1, 200:1, 100:0) on the reversal of biochemical EFAD. Results Mice provided with DHA and AA had no evidence of biochemical EFAD, regardless of the ratio (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) administered. Biochemical EFAD was reversed with DHA:AA ratios of 20:1, 200:1, and 100:0 following 3 and 5 weeks of dietary provision, although the 20:1 ratio was most effective in the reversal and stabilization of the triene:tetraene ratio. Conclusion Provision of DHA and AA, at 2.1% of daily caloric intake in varying ratios can prevent biochemical evidence of EFAD and hepatic steatosis over the short-term, with a ratio of 20:1 DHA:AA most effectively reversing EFAD. PMID:23151438

  12. Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function.

    PubMed Central

    Ritsema, T; Geiger, O; van Dillewijn, P; Lugtenberg, B J; Spaink, H P

    1994-01-01

    A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed. We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional. Images PMID:8002602

  13. Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.

    PubMed

    Liu, Dan; Zhang, Zheng; Liman, Emily R

    2005-05-27

    TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

  14. Essential fatty acids prevent slowed nerve conduction in streptozotocin diabetic rats.

    PubMed

    Julu, P O

    1988-01-01

    Rats were given streptozotocin to induce insulin-dependent diabetes or citrate buffer alone in two experiments. Initially, the effect of 5 wks of dietary gamma-linolenic acid (GLA) plus eicosapentaenoic acid (EPA) on cutaneous nerve conduction velocity (CV) was examined. CV was determined by direct stimulation and recording from saphenous nerve under urethane anesthesia. Secondly, a 5 weeks study of supplementing the diet with GLA, GLA and EPA, or hydrogenated coconut oil (HC) was done. In addition, motor nerve CV was determined by directly stimulating sciatic nerve and recording from gastrocnemius muscle. The acute diabetes led to weight loss, and elevated blood glucose and glycosylated hemoglobin levels. Essential fatty acid (EFA) supplementation had no effect on any of these measures of severity of diabetes. In diabetic rats without EFA supplementation, CV of the myelinated fibers fell by 19-21%, while those receiving both GLA and EPA had normal CV. In diabetic rats receiving GLA alone, CV fell by 5-7%, which was significantly less than those without EFA supplementation (p less than 0.01 for cutaneous, and p less than 0.001 for motor nerves).

  15. 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

  16. 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

  17. 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

  18. Does zinc moderate essential fatty acid and amphetamine treatment of attention-deficit/hyperactivity disorder?

    PubMed

    Arnold, L E; Pinkham, S M; Votolato, N

    2000-01-01

    Zinc is an important co-factor for metabolism relevant to neurotransmitters, fatty acids, prostaglandins, and melatonin, and indirectly affects dopamine metabolism, believed intimately involved in attention-deficit/hyperactivity disorder (ADHD). To explore the relationship of zinc nutrition to essential fatty acid supplement and stimulant effects in treatment of ADHD, we re-analyzed data from an 18-subject double-blind, placebo-controlled crossover treatment comparison of d-amphetamine and Efamol (evening primrose oil, rich in gamma-linolenic acid). Subjects were categorized as zinc-adequate (n = 5), borderline zinc (n = 5), and zinc-deficient (n = 8) by hair, red cell, and urine zinc levels; for each category, placebo-active difference means were calculated on teachers' ratings. Placebo-controlled d-amphetamine response appeared linear with zinc nutrition, but the relationship of Efamol response to zinc appeared U-shaped; Efamol benefit was evident only with borderline zinc. Placebo-controlled effect size (Cohen's d) for both treatments ranged up to 1.5 for borderline zinc and dropped to 0.3-0.7 with mild zinc deficiency. If upheld by prospective research, this post-hoc exploration suggests that zinc nutrition may be important for treatment of ADHD even by pharmacotherapy, and if Efamol benefits ADHD, it likely does so by improving or compensating for borderline zinc nutrition. PMID:10933121

  19. 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.

  20. Zinc-Mediated Binding of Nucleic Acids to Amyloid-β Aggregates: Role of Histidine Residues.

    PubMed

    Khmeleva, Svetlana A; Radko, Sergey P; Kozin, Sergey A; Kiseleva, Yana Y; Mezentsev, Yuri V; Mitkevich, Vladimir A; Kurbatov, Leonid K; Ivanov, Alexis S; Makarov, Alexander A

    2016-09-01

    Amyloid-β peptide (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aβ, intraneuronal Aβ (iAβ) has been suggested to contribute to AD onset and development. Based on reported in vitro Aβ-DNA interactions and nuclear localization of iAβ, the interference of iAβ with the normal DNA expression has recently been proposed as a plausible pathway by which Aβ can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aβ42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed β-sheet rich Aβ42 aggregates. Another type of Aβ42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aβ metal-binding domain's histidine residues in Aβ-nucleic acid interactions mediated by zinc, Aβ16 mutants with substitutions H6R and H6A-H13A and rat Aβ16 lacking histidine residue 13 were used. The zinc-induced interaction of Aβ16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aβ42 peptide did not affect DNA binding to Aβ42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAβ-nucleic acid interactions. PMID:27567853

  1. 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.

  2. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

    PubMed

    Sayanova, O; Shewry, P R; Napier, J A

    1999-10-01

    Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

  3. Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity1

    PubMed Central

    Sayanova, Olga; Shewry, Peter R.; Napier, Johnathan A.

    1999-01-01

    Unlike most other plant microsomal desaturases, the Δ6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Δ6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ6-fatty acid desaturase resulted in the synthesis and accumulation of Δ6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Δ6-desaturase in transgenic plants failed to produce Δ6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Δ6-fatty acid desaturase is essential for enzymatic activity. PMID:10517856

  4. 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

  5. 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

  6. Increasing dietary crude protein does not increase the essential amino acid requirements of kittens.

    PubMed

    Strieker, M J; Morris, J G; Rogers, Q R

    2006-08-01

    Essential amino acid (EAA) requirements of omnivores and herbivores (e.g. chicks, lambs, pigs and rats) are directly related to the concentration of dietary crude protein (CP). When an EAA is limiting in the diet, addition of a mixture of EAA lacking the limiting one (which increases dietary CP) results in a decrease in food intake and weight gain. This interaction has been referred to as an AA imbalance and has not been studied in depth in strict carnivores. The objectives of these experiments were to examine the effects on growing kittens (2-week periods) of the addition to diets of a mixture of AA lacking the limiting one. The control diets were at the requirement of the respective limiting EAA (or about 85% of the 1986 National Research Council requirement). In experiment 1, with the dietary EAAs at the minimally determined requirements, the concentration of the essential or dispensable amino acids was increased to determine if CP or an EAA was limiting. Results of growth rates (n = 12) and plasma AA concentrations indicated that tryptophan was limiting, but increased body weight gain also occurred when the concentration of CP was increased as dispensable amino acids without additional tryptophan. Experiment 1 was repeated in experiment 2 using a crossover design. Again, when tryptophan was limiting additional concentrations of dispensable AAs increased body weight gain. This response is the opposite of that in herbivores and omnivores. Experiment 3 consisted of 10 separate crossover trials, one for each of the 10 EAA and examined the effect of two concentrations of dietary CP (200 and 300 g CP/kg diet) on body weight gain of kittens (n = 8) offered diets limiting in each respective EAA. Body weight gain was numerically greater when diets contained 300 g CP/kg than 200 g CP/kg for eight of 10 EAAs (p < 0.05 for only isoleucine and threonine) when each amino acid was limiting. This response is the reverse of that which occurs in chicks, lambs, pigs and rats when

  7. Infectious tolerance via the consumption of essential amino acids and mTOR signaling.

    PubMed

    Cobbold, Stephen P; Adams, Elizabeth; Farquhar, Claire A; Nolan, Kathleen F; Howie, Duncan; Lui, Kathy O; Fairchild, Paul J; Mellor, Andrew L; Ron, David; Waldmann, Herman

    2009-07-21

    Infectious tolerance describes the process of CD4(+) regulatory T cells (Tregs) converting naïve T cells to become additional Tregs. We show that antigen-specific Tregs induce, within skin grafts and dendritic cells, the expression of enzymes that consume at least 5 different essential amino acids (EAAs). T cells fail to proliferate in response to antigen when any 1, or more, of these EAAs are limiting, which is associated with a reduced mammalian target of rapamycin (mTOR) signaling. Inhibition of the mTOR pathway by limiting EAAs, or by specific inhibitors, induces the Treg-specific transcription factor forkhead box P3, which depends on both T cell receptor activation and synergy with TGF-beta. PMID:19567830

  8. [The use of essential fatty acids in the treatments of wounds].

    PubMed

    Manhezi, Andreza Cano; Bachion, Maria Márcia; Pereira, Angela Lima

    2008-01-01

    In spite of being widely spread throughout Brazil, the use of essential fatty acids (EFA) for wound healing is controversial. This study aimed at identifying and analyzing the available scientific evidence for EFA to be used in the treatment of wounds. This is a descriptive study, carried out through a systematic literature review, concerning the Biblioteca Virtual de Saúde (Health Online Library) and PubMed data bank, from 1970 to 2006. Initially, we identified 503 references. After the relevance tests I and II, 11 articles were included in the analysis, showing evidence of recommendation- level II and III for EFA to be used in burns, mediastinitis, among others situations. Most studies still refer to its use in animal. Relevant publications are still scarce.

  9. A Nitrogen-concentrated Phase in IA Iron Meteorite Acid Residue

    NASA Astrophysics Data System (ADS)

    Hashizume, K.; Sugiura, N.

    1993-07-01

    Introduction: Iron meteorites are considered to have experienced a complex history, which is indicated by the variations in trace element chemistry (e.g., [1]). Among iron meteorite groups, the so called nonmagmatic groups, such as IAB, IIE, and IIICD, may have passed through different formation paths compared to others. Nitrogen isotopes can be a useful tool to understand the origin and formation processes of iron meteorites. Nikogen isotopes in a number of iron meteorites are measured [2,3], although trapping sites of nitrogen in iron meteorites are not yet clear. This is an important issue because nitrogen, a typical mobile element, may well reflect thermal history of their parent bodies (c.f., [4]). Generally, a major portion of nitrogen in iron meteorites is expected to be in a solid solution in Fe-Ni, especially in f.c.c. Fe-Ni (taenite). Franchi et al. [3] report that at least 25 to 35% of nitrogen in magmatic iron meteorites is in acid insoluble phases, however, not in those of non-magmatic meteorites. This result contradicts with the result [5] who report that a significant portion of nitrogen seems to be trapped in acid residues not only of magmatic meteorites but also of non- magmatic meteorites. To resolve the contradiction described above, and to identify the trapping site, we started measuring nitrogen isotopes in acid residues of iron metcorites. We report here preliminary results on acid residues of Canyon Diablo (IA). Procedures: Acid residues were prepared by Dr. J.-I. Matsuda and his colleagues. Different blocks of Canyon Diablo, "Can-1" and "Can-2" were treated by 14M HCl, 10M-HF + 1M-HCl, 1M-HCl, and by aqua regia, which destroyed Fe-Ni, sulfides, silicates, and shreibersite. Acid residues of these two blocks, "Can-1bn" and "Can-2b," yielded 0.102 wt% and 0.299 wt% of their original masses, respectively These residues seem to consist mostly of graphite No diamond was detected by powder X-ray analysis [6]. Preliminary Results: A predominant

  10. 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.

  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. Differentiating amino acid residues and side chain orientations in peptides using scanning tunneling microscopy.

    PubMed

    Claridge, Shelley A; Thomas, John C; Silverman, Miles A; Schwartz, Jeffrey J; Yang, Yanlian; Wang, Chen; Weiss, Paul S

    2013-12-11

    Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.

  14. Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues.

    PubMed

    Zhang, Qinghua; Tang, Lei; Zhang, Jianhua; Mao, Zhonggui; Jiang, Li

    2011-02-01

    In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H(2)SO(4) concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84°C, utilizing 2.99% (w/w TS) H(2)SO(4) for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.

  15. Residues Essential for Panton-Valentine Leukocidin S Component Binding to Its Cell Receptor Suggest Both Plasticity and Adaptability in Its Interaction Surface

    PubMed Central

    Laventie, Benoit-Joseph; Guérin, Frédéric; Mourey, Lionel; Tawk, Mira Y.; Jover, Emmanuel; Maveyraud, Laurent; Prévost, Gilles

    2014-01-01

    Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10−10 M) compared to the class F component of PVL, LukF-PV (Kd∼10−9 M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV. PMID:24643034

  16. Effects of the number of fatty acid residues on the phase behaviors of decaglycerol fatty acid esters.

    PubMed

    Ai, Sakiko; Ishitobi, Masahiko

    2006-04-15

    The effects of the number of fatty acid residues (n) in decaglycerol fatty acid esters, i.e., decaglycerol laurates (abbreviated to (C11)nG10), on the phase behaviors of three laurate esters, (C11)1.9G10, (C11)2.7G10, and (C11)3.4G10, were investigated. The unreacted decaglycerol remaining in each ester was removed by liquid extraction before use. (C11)1.9G10 formed hexagonal liquid crystals in aqueous solutions, while (C11)2.7G10 and (C11)3.4G10, which are more hydrophobic than (C11)1.9G10, formed lamellar liquid crystals. The cloud point in aqueous solution was measured for mixtures of these three esters. The cloud phenomenon was observed when the weight ratio of hydrophilic groups to the total surfactant (WH/WS) was around 0.6. The cloud point shifted to a markedly higher temperature, even with a slight increase in the WH/WS ratio. The solubilization abilities of (C11)nG10 for the oils m-xylene and (R)-(+)-limonene were also examined. When the WH/WS ratio was between 0.60 and 0.64, (C11)nG10 formed microemulsions and lyotropic liquid crystals in the presence of water and the oils. These self-organized structures were stable, even above 90 degrees C. It is concluded that the phase behavior of (C11)nG10 are insensitive to temperature, but strongly dependent on both the WH/WS ratio and the number of fatty acid residues (n).

  17. Solid substrate fermentation of cassava fibrous residue for production of alpha-amylase, lactic acid and ethanol.

    PubMed

    Ray, Ramesh C; Mohapatra, Sabita; Panda, Shrutirupa; Kar, Shaktimay

    2008-01-01

    There is serious concern about the disposal of solid residues left after large scale extraction of starch from cassava. Owing to the high starch content (55-65% on dry weight basis) and organic matter of these wastes, an attempt has been made to utilize it for the production of three bioproducts, i.e. alpha-amylase, lactic acid and ethanol in solid substrate fermentation by incubating the solid residue at different moisture holding capacity (40-80%) and incubation period (12- 60 hr for alpha-amylase, 24-144 hr for ethanol and 2-10 days for lactic acid). The highest product yield was obtained at 60% moisture holding capacity of the residue and period of incubation varied from 36 hr (alpha-amylase), 120 hr (ethanol) to 6 days (lactic acid). This study showed that the solid residues from cassava starch factories could serve as a low-cost substrate for bioproducts production.

  18. Development of an in vitro model of essential fatty acid deficiency in fish cells.

    PubMed

    Tocher, D R; Dick, J R; Sargent, J R

    1995-11-01

    Levels of eicosapentaenoic acid (EPA; 20:5, n-3) greatly exceed those of arachidonic acid (AA; 20:4, n-6) in the tissue phospholipids of most fish species. Despite this, it is 20:4, n-6-derived eicosanoids that are produced predominantly in fish cells. The development of an essential fatty acid (EFA)-deficient fish cell line would greatly assist the study of this selectivity and so several fish cell lines were cultured in EFA-deficient (EFAD) media. All n-3 and n-6 polyunsaturated fatty acids (PUFA) and total PUFA were considerably reduced in all lines, except turbot fin (TF) in which total n-9 PUFA doubled from 13.8% to 27.5% of total fatty acids. In the topminnow hepatocarcinoma cell line (PLHC-1), there was almost complete depletion of both n-3 and n-6 PUFA and in TF cells, no n-3 PUFA were detected. In the carp epithelial papilloma cell line (EPC), both n-6 and n-3 PUFA were reduced by approximately 70%. The reduced PUFA in cells cultured in EFAD media was compensated to a large extent in most cell lines by significantly increased percentages of monounsaturated fatty acids, particularly 18:1, n-9. Total n-9 PUFA were significantly increased in all cell lines by culture in EFAD media, with 20:2, n-9 significantly increased in all cell lines. There were relatively small increases, but often significant, in 20:3, n-9 in all cell lines. Of the cell lines investigated, only EPC and PLHC-1 showed proliferation after four passages in EFAD medium, although the growth rates were reduced in comparison with media supplemented with serum, but EPC was the only cell line able to survive and proliferate in long-term culture on EFAD medium. The EFAD-EPC line is a potentially useful model system for the study of the effects of EFA deficiency on cell structure and function and eicosanoid metabolism in fish. PMID:8596777

  19. Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

    PubMed

    Toal, Siobhan E; Verbaro, Daniel J; Schweitzer-Stenner, Reinhard

    2014-02-01

    The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

  20. 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

  1. 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

  2. Glycolic acid-catalyzed deamidation of asparagine residues in degrading PLGA matrices: a computational study.

    PubMed

    Manabe, Noriyoshi; Kirikoshi, Ryota; Takahashi, Ohgi

    2015-03-31

    Poly(lactic-co-glycolic acid) (PLGA) is a strong candidate for being a drug carrier in drug delivery systems because of its biocompatibility and biodegradability. However, in degrading PLGA matrices, the encapsulated peptide and protein drugs can undergo various degradation reactions, including deamidation at asparagine (Asn) residues to give a succinimide species, which may affect their potency and/or safety. Here, we show computationally that glycolic acid (GA) in its undissociated form, which can exist in high concentration in degrading PLGA matrices, can catalyze the succinimide formation from Asn residues by acting as a proton-transfer mediator. A two-step mechanism was studied by quantum-chemical calculations using Ace-Asn-Nme (Ace = acetyl, Nme = NHCH3) as a model compound. The first step is cyclization (intramolecular addition) to form a tetrahedral intermediate, and the second step is elimination of ammonia from the intermediate. Both steps involve an extensive bond reorganization mediated by a GA molecule, and the first step was predicted to be rate-determining. The present findings are expected to be useful in the design of more effective and safe PLGA devices.

  3. 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

  4. 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

  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%).

  6. 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

  7. 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

  8. 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

  9. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  10. Acid-catalyzed hydrothermal severity on the fractionation of agricultural residues for xylose-rich hydrolyzates.

    PubMed

    Lee, Ji Ye; Ryu, Hyun Jin; Oh, Kyeong Keun

    2013-03-01

    The objective of this work was to investigate the feasibility of acid-catalyzed hydrothermal fractionation for maximum solubilization of the hemicellulosic portion of three agricultural residues. The fractionation conditions converted into combined severity factor (CS) in the range of 1.2-2.9. The highest hemicellulose yield of 87.88% was achieved when barley straw was fractionated at a CS of 2.19. However, the maximum glucose release of 15.29% was achieved for the case of rice straw. The maximum productions of various by-products were observed with the fractionation of rape straw: 0.88 g/L of 5-hydroxymethylfurfural (5-HMF), 2.16 g/L of furfural, 0.44 g/L of levulinic acid, 1.59 g/L of formic acid, and 3.06 g/L of acetic acid. The highest selectivities, a criterion for evaluating the fractionation of 21.55 for fractionated solid and 7.48 for liquid hydrolyzate were obtained from barley straw.

  11. Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum.

    PubMed

    McMillan, Paul J; Arscott, L David; Ballou, David P; Becker, Katja; Williams, Charles H; Müller, Sylke

    2006-11-01

    High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

  12. 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.

  13. Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.

    PubMed

    Wang, Jing; Rabenstein, Dallas L

    2007-09-01

    Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.

  14. 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

  15. 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.

  16. 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

  17. New force field parameters for metalloproteins I: Divalent copper ion centers including three histidine residues and an oxygen-ligated amino acid residue.

    PubMed

    Wise, Olivia; Coskuner, Orkid

    2014-06-30

    Transition metal ion complexation with proteins is ubiquitous across such diverse fields as neurodegenerative and cardiovascular diseases and cancer. In this study, the structures of divalent copper ion centers including three histidine and one oxygen-ligated amino acid residues and the relative binding affinities of the oxygen-ligated amino acid residues with these metal ion centers, which are debated in the literature, are presented. Furthermore, new force field parameters, which are currently lacking for the full-length metal-ligand moieties, are developed for metalloproteins that have these centers. These new force field parameters enable investigations of metalloproteins possessing these binding sites using molecular simulations. In addition, the impact of using the atom equivalence and inequivalence atomic partial charge calculation procedures on the simulated structures of these metallopeptides, including hydration properties, is described.

  18. 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

  19. Using 3–6 differences in essential fatty acids rather than 3/6 ratios gives useful food balance scores

    PubMed Central

    2012-01-01

    Background The vitamin-like omega-3 and omega-6 essential fatty acids are converted in the body to a large family of hormones which act at selective receptors that occur on nearly every cell and tissue. A relative omega-3 deficit allows overabundant actions of omega-6 hormones to develop into health disorders. People need simple, explicit information on the balance of essential fatty acids in their foods to avoid accumulating unintended imbalances in their tissue omega-3 and omega-6 fatty acids. Results We developed an Omega 3–6 Balance Food Score that summarizes in a single value the balance among eleven omega-3 and omega-6 essential fatty acids in a food. The value allows a quantitative estimate of the impact of each food item on the proportions of omega-3 and omega-6 that will accumulate in the 20- and 22-carbon highly unsaturated fatty acids of blood, which is an important health risk assessment biomarker. Conclusions The impact of an individual food item upon a useful health risk assessment biomarker is easily evident in a simple, explicit value for the balance among eleven essential fatty acids nutrients. Foods with more positive Omega 3–6 Balance Food Scores will increase the percent of omega-3 in the biomarker, whereas those with more negative Scores will increase the percent of omega-6 in the biomarker. PMID:22624598

  20. 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.

  1. 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

  2. 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.

  3. Effects of essential fatty acid deficiency on enterohepatic circulation of bile salts in mice.

    PubMed

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

    2009-09-01

    Essential fatty acid (EFA) deficiency in mice has been associated with increased bile production, which is mainly determined by the enterohepatic circulation (EHC) of bile salts. To establish the mechanism underlying the increased bile production, we characterized in detail the EHC of bile salts in EFA-deficient mice using stable isotope technique, without interrupting the normal EHC. Farnesoid X receptor (FXR) has been proposed as an important regulator of bile salt synthesis and homeostasis. In Fxr(-/-) mice we additionally investigated to what extent alterations in bile production during EFA deficiency were FXR dependent. Furthermore, we tested in differentiating Caco-2 cells the effects of EFA deficiency on expression of FXR-target genes relevant for feedback regulation of bile salt synthesis. EFA deficiency-enhanced bile flow and biliary bile salt secretion were associated with elevated bile salt pool size and synthesis rate (+146 and +42%, respectively, P < 0.05), despite increased ileal bile salt reabsorption (+228%, P < 0.05). Cyp7a1 mRNA expression was unaffected in EFA-deficient mice. However, ileal mRNA expression of Fgf15 (inhibitor of bile salt synthesis) was significantly reduced, in agreement with absent inhibition of the hepatic bile salt synthesis. Bile flow and biliary secretion were enhanced to the same extent in EFA-deficient wild-type and Fxr(-/-) mice, indicating contribution of other factors besides FXR in regulation of EHC during EFA deficiency. In vitro experiments show reduced induction of mRNA expression of relevant genes upon chenodeoxycholic acid and a selective FXR agonist GW4064 stimulation in EFA-deficient Caco-2 cells. In conclusion, our data indicate that EFA deficiency is associated with interrupted negative feedback of bile salt synthesis, possibly because of reduced ileal Fgf15 expression.

  4. 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

  5. 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

  6. 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

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

    PubMed Central

    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

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  10. 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

  11. Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.

    PubMed

    Niederreither, K; Subbarayan, V; Dollé, P; Chambon, P

    1999-04-01

    A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death. PMID:10192400

  12. 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

  13. Nutritional balance of essential amino acids and carbohydrates of the adult worker honeybee depends on age.

    PubMed

    Paoli, Pier P; Donley, Dion; Stabler, Daniel; Saseendranath, Anumodh; Nicolson, Susan W; Simpson, Stephen J; Wright, Geraldine A

    2014-06-01

    Dietary sources of essential amino acids (EAAs) are used for growth, somatic maintenance and reproduction. Eusocial insect workers such as honeybees are sterile, and unlike other animals, their nutritional needs should be largely dictated by somatic demands that arise from their role within the colony. Here, we investigated the extent to which the dietary requirements of adult worker honeybees for EAAs and carbohydrates are affected by behavioural caste using the Geometric Framework for nutrition. The nutritional optimum, or intake target (IT), was determined by confining cohorts of 20 young bees or foragers to liquid diets composed of specific proportions of EAAs and sucrose. The IT of young, queenless bees shifted from a proportion of EAAs-to-carbohydrates (EAA:C) of 1:50 towards 1:75 over a 2-week period, accompanied by a reduced lifespan on diets high in EAAs. Foragers required a diet high in carbohydrates (1:250) and also had low survival on diets high in EAA. Workers exposed to queen mandibular pheromone lived longer on diets high in EAA, even when those diets contained 5× their dietary requirements. Our data show that worker honeybees prioritize their intake of carbohydrates over dietary EAAs, even when overeating EAAs to obtain sufficient carbohydrates results in a shorter lifespan. Thus, our data demonstrate that even when young bees are not nursing brood and foragers are not flying, their nutritional needs shift towards a diet largely composed of carbohydrates when they make the transition from within-hive duties to foraging.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide

    PubMed Central

    Choi, Byung-Kwon; Schifferli, Dieter M.

    1999-01-01

    The FasG subunit of the 987P fimbriae of enterotoxigenic strains of Escherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in four fasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction. PMID:10531225

  19. 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).

  20. Trace analysis of acidic pharmaceutical residues in waters with isotope dilution gas chromatography-mass spectrometry via methylation derivatization.

    PubMed

    Hu, Ruikang; Yang, Zhaoguang; Zhang, Lifeng

    2011-09-30

    Acidic pharmaceutical residues are pollutants of emerging concern and are generally monitored by HPLC-MS/MS. However, due to the limited separation efficiency of HPLC column and lack of suitable mass transition for confirmation analysis, some interference may not be separated completely and differentiated from ibuprofen, which may cause the results with interference, especially in sample with complex matrix. The objective of this study is to develop a sensitive and reliable method for the determination of acidic pharmaceutical residues in water samples by GC-MS with better resolution by using methylation derivatization and isotope dilution techniques. TMSDM, a mild reagent, was used as the derivatization reagent coupling with the isotope dilution technique, for the first time, to improve the precision and accuracy of the analytical method to determine the pharmaceutical residues in water. The MDLs for the five acidic organic compounds: ibuprofen, gemfibrozil, naproxen, ketoprofen and diclofenac were from 0.7 to 1.1 ng/L, with recoveries ranging from 93 to 110%. Alternative to the HPLC-MS/MS method, the developed GC-MS protocols provides an additional option for the analysis of acidic pharmaceutical residues in water, with better separation efficiency in reducing interferences from complicated sample matrix, for determination of ibuprofen residues.

  1. Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase.

    PubMed

    Keynan, S; Hooper, N M; Turner, A J

    1997-08-15

    Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

  2. Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

    PubMed

    Keskar, S S; Srinivasan, M C; Deshpande, V V

    1989-07-01

    Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

  3. Amino-acid residues involved in the expression of the activity of Escherichia coli TolC.

    PubMed

    Yamanaka, Hiroyasu; Morisada, Naoyuki; Miyano, Masaya; Tsuge, Hideaki; Shinoda, Sumio; Takahashi, Eizo; Okamoto, Keinosuke

    2004-01-01

    The Escherichia coli TolC, composed of 471 amino-acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu-412, the 60th amino-acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu-412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu-412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81-89, 2002). We predicted that other hydrophobic amino-acid residues around Leu-412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu-412, (Leu-3, Val-6, Leu-212, Leu-213, Leu-223, and Leu-224), with serine and examined the activity of these mutant TolCs. The result showed that Leu-3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu-3 was confirmed by the residue deletion experiment. A subsequent point-mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the alpha-carbons of Leu-3 and Leu-412 is just 7.45 angstroms, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC. PMID:15502403

  4. 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

  5. Experimental test of the superheavy fission hypothesis in acid residues from the allende meteorite

    SciTech Connect

    Flynn, G.J.

    1982-01-01

    A description of a series of experiments to find evidence to confirm or contradict the hypothesis that isotopically anomalous Xe (called CCF-Xe) in carbonaceous chondrite meteorites results from the fission decay of a superheavy element is given. The first two experiments were searches for fossil evidence - fission tracks and isotopic anomalies - of superheavy fission decay in the Allende carbonaceous chondrite. It was demonstrated that chromite, a mineral rich in CCF-Xe, records fission tracks, and a search for such tracks in Allende chromite was performed with negative results. It was also demonstrated in certain CCF-XE rich phases of Allende, isotopic anomalies like those seen in Xe should be detectable in Ba and the light rare earths. Preliminary results from a collaborative measurement (with the University of Paris) show the Ba isotopic ratios to be normal in a CCF-Xe rich Allende sample. The third experiment was motivated by reports of the detection of a live, fissioning superheavy element, using a neutron counting technique, in bulk Allende (Flerov, 1978). Since almost all of the allegedly fissiogenic Xe in Allende is concentrated in certain acid insoluble phases, we developed a technique to detect low-level fission activity in these phases. Allende acid insoluble residue (provided by the University of Chicago) was dispersed in a 1 mg/cm/sup 2/ layer, between two track recording detectors. An automatic track locating system was developed to allow large detector areas to be scanned for rate fission events.

  6. 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

  7. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  8. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  9. Stimulation of proliferation of an essential fatty acid-deficient fish cell line by C20 and C22 polyunsaturated fatty acids and effects on fatty acid composition.

    PubMed

    Tocher, D R; Dick, J R; Sargent, J R

    1996-11-01

    Recently we reported the development of a fish cell line, EPC-EFAD, derived from the carp (Cyprinus carpio) epithelial papilloma line, EPC, that could survive and proliferate in essential fatty acid-deficient (EFAD) medium. The EPC-EFAD cell line may be a useful model system in which to study the cellular biochemical effects of EFA deficiency and has advantages in studies of polyunsaturated fatty acid (PUFA) and eicosanoid metabolism in fish in that the complications introduced by culture in relatively n-6 PUFA-rich mammalian sera are removed. In the present study, the effects on cell proliferation rate of supplementing EPC-EFAD cells with various n-3 and n-6 PUFA were investigated to determine the possible role(s) of PUFA in cell growth and division. The selectivity of incorporation of specific PUFA into individual glycerophospholipid classes and the feasibility of reproducing in vivo fatty acid compositions in vitro were also investigated. Proliferation of the EPC-EFAD cell line was stimulated by arachidonic (20:4 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) fatty acids but not by 18:2 n-6 or 18:3 n-3. The differential effects of PUFA on cellular proliferation may be related to the lack of significant delta 5 desaturase activity in the cells at 22 degrees C and may implicate a role for eicosanoids in the mechanism of stimulation of proliferation. PUFA supplementation increased the cytotoxic effects of longer term culture, an effect that was partly alleviated by inclusion of vitamin E in the culture medium. The cells could generally be supplemented with PUFA to produce cellular fatty acid compositions in vitro that were similar to in vivo compositions. PMID:8981632

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Nutritional balance of essential amino acids and carbohydrates of the adult worker honeybee depends on age.

    PubMed

    Paoli, Pier P; Donley, Dion; Stabler, Daniel; Saseendranath, Anumodh; Nicolson, Susan W; Simpson, Stephen J; Wright, Geraldine A

    2014-06-01

    Dietary sources of essential amino acids (EAAs) are used for growth, somatic maintenance and reproduction. Eusocial insect workers such as honeybees are sterile, and unlike other animals, their nutritional needs should be largely dictated by somatic demands that arise from their role within the colony. Here, we investigated the extent to which the dietary requirements of adult worker honeybees for EAAs and carbohydrates are affected by behavioural caste using the Geometric Framework for nutrition. The nutritional optimum, or intake target (IT), was determined by confining cohorts of 20 young bees or foragers to liquid diets composed of specific proportions of EAAs and sucrose. The IT of young, queenless bees shifted from a proportion of EAAs-to-carbohydrates (EAA:C) of 1:50 towards 1:75 over a 2-week period, accompanied by a reduced lifespan on diets high in EAAs. Foragers required a diet high in carbohydrates (1:250) and also had low survival on diets high in EAA. Workers exposed to queen mandibular pheromone lived longer on diets high in EAA, even when those diets contained 5× their dietary requirements. Our data show that worker honeybees prioritize their intake of carbohydrates over dietary EAAs, even when overeating EAAs to obtain sufficient carbohydrates results in a shorter lifespan. Thus, our data demonstrate that even when young bees are not nursing brood and foragers are not flying, their nutritional needs shift towards a diet largely composed of carbohydrates when they make the transition from within-hive duties to foraging. PMID:24623119

  15. 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

  16. Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos.

    PubMed

    Li, Junbo; Yue, Yunyun; Zhao, Qingshun

    2016-02-01

    Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

  17. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    PubMed

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system. PMID:25000760

  18. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    PubMed

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system.

  19. Effects of essential fatty acid deficiency on epidermal O-acylsphingolipids and transepidermal water loss in young pigs.

    PubMed

    Melton, J L; Wertz, P W; Swartzendruber, D C; Downing, D T

    1987-09-25

    Linoleate-rich O-acylglucosylceramides and acylceramides are thought to be of major significance for the physical structure and function of the epidermal permeability barrier. In the present investigation, the effects of a linoleate-free diet on O-acylsphingolipids and their associated functions were investigated. Starting at 5 days of age, male pigs were fed diets containing 12% of either lard or hydrogenated coconut oil. Transepidermal water loss was measured with an electrolytic water analyzer at weekly intervals. Pigs were killed at intervals, and epidermal lipids were isolated and analyzed. Fatty acid compositions were determined by gas-liquid chromatography (GLC). Within 2-3 weeks, pigs on the diet containing coconut oil began to display biochemical and physiological symptoms of essential fatty acid deficiency. Within 2 months, this group had extremely scaly skin and transepidermal water loss was elevated to five times that of controls. The progressive increase in transepidermal water loss correlated with replacement of linoleate by oleate in both acylceramide and acylglucosylceramide. The formation of lamellar granules and intercellular lipid sheets in the stratum corneum was not impaired in essential fatty acid deficiency as judged by electron microscopy. These results suggest that the linoleic acid normally found in the O-acylsphingolipids is not essential for formation of the epidermal membrane system. Rather, it appears that the nature of the ester-linked fatty acid in the O-acylsphingolipids regulates the permeability of this membrane system.

  20. Fibrates are an essential part of modern anti-dyslipidemic arsenal: spotlight on atherogenic dyslipidemia and residual risk reduction.

    PubMed

    Tenenbaum, Alexander; Fisman, Enrique Z

    2012-10-11

    glucose metabolism and insulin resistance. Bezafibrate is the only clinically available pan - (alpha, beta, gamma) PPAR balanced activator. Bezafibrate decreases blood glucose level, HbA1C, insulin resistance and reduces the incidence of T2DM compared to placebo or other fibrates. Among major fibrates, bezafibrate appears to have the strongest and fenofibrate the weakest effect on HDL-C. Current therapeutic use of statins as monotherapy is still leaving many patients with atherogenic dyslipidemia at high risk for coronary events because even intensive statin therapy does not eliminate the residual cardiovascular risk associated with low HDL and/or high triglycerides. As compared with statin monotherapy (effective mainly on LDL-C levels and plaque stabilization), the association of a statin with a fibrate will also have a major impact on triglycerides, HDL and LDL particle size. Moreover, in the specific case of bezafibrate one could expect neutralizing of the adverse pro-diabetic effect of statins. Though muscle pain and myositis is an issue in statin/fibrate treatment, adverse interaction appears to occur to a significantly greater extent when gemfibrozil is administered. However, bezafibrate and fenofibrate seems to be safer and better tolerated. Combined fibrate/statin therapy is more effective in achieving a comprehensive lipid control and may lead to additional cardiovascular risk reduction, as could be suggested for fenofibrate following ACCORD Lipid study subgroup analysis and for bezafibrate on the basis of one small randomized study and multiple observational data. Therefore, in appropriate patients with atherogenic dyslipidemia fibrates- either as monotherapy or combined with statins - are consistently associated with reduced risk of cardiovascular events. Fibrates currently constitute an indispensable part of the modern anti-dyslipidemic arsenal for patients with atherogenic dyslipidemia.

  1. Ruminal degradation, amino acid composition, and intestinal digestibility of the residual components of five protein supplements.

    PubMed

    Maiga, H A; Schingoethe, D J; Henson, J E

    1996-09-01

    Two ruminally cannulated Holstein cows (approximately 202 DIM) were used to determine the in situ degradability of five protein supplements: blood meal, meat and bone meal, corn gluten meal, expeller soybean meal, and solvent extracted soybean meal. Dacron bags containing 4 g of each supplement in duplicate were soaked in water and then incubated in the rumen for 0, 3, 6, 12, 18, and 24 h for 3 d. Four extra sample bags of each supplement were incubated in the rumen for 12 h to determine the in vitro intestinal digestibility and AA analysis of the residues. Protein supplements were also analyzed for their AA content. Ruminal degradability of individual supplements varied. Solvent soybean meal was the most degradable, and blood meal was the least degradable. Specific first-limiting essential AA were isoleucine for blood meal and meat and bone meal, lysine for corn gluten meal, and methionine for the soybean meals. The RUP fraction in solvent-extracted and expeller soybean meals tended to be more intestinally digestible than did the protein in blood meal and meat and bone meal. In general, all protein supplements, except solvent-extracted soybean meal, were high in RUP and had the potential to provide good quality AA to complement microbial AA for production.

  2. 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

  3. Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.

    PubMed

    Dmitrieva, Tatiana M; Alexeevski, Andrei V; Shatskaya, Galina S; Tolskaya, Elena A; Gmyl, Anatoly P; Khitrina, Elena V; Agol, Vadim I

    2007-08-15

    Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed. PMID:17467026

  4. Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11.

    PubMed

    Le Moual, H; Devault, A; Roques, B P; Crine, P; Boileau, G

    1991-08-25

    Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.

  5. 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.

  6. 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

  7. 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

  8. Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine.

    PubMed

    Lee, Y S; Cho, Y D

    2001-12-15

    The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC

  9. 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

  10. Effect of low molecular weight organic acids on phosphorus adsorption by ferric-alum water treatment residuals.

    PubMed

    Wang, Changhui; Wang, Ziyuan; Lin, Lu; Tian, Binghui; Pei, Yuansheng

    2012-02-15

    Effects of low molecular weight organic acids (LMWOAs; citric acid, oxalic acid and tartaric acid) on phosphorus (P) adsorption by ferric-alum water treatment residuals (FARs) were studied. Both batch and column experiments indicated that the effects of LMWOAs on P adsorption were closely related to adsorption time. Initially, all acids presented inhibitory function on P adsorption. The inhibition became weaker with time, eventually promoting P adsorption for citric acid and tartaric acid. In the column experiment with a 61-day duration, high P adsorption rates (>55%) were observed for the test groups containing citric acid and tartaric acid. Interestingly, higher pH likely enhanced P adsorption with the effects of LMWOAs and a distinct relationship between LMWOAs' effects on P adsorption and their concentrations was not observed. Moreover, fractionation of the adsorbed P from the FARs demonstrated that oxalic acid reduced P adsorption capacity, while citric acid and tartaric acid increased. Based on the forms of Fe and Al existing in the FARs and Fourier transform infrared spectroscopy analyses, LMWOAs can promote P adsorption through activating crystalline Fe/Al and preventing crystallization of amorphous Fe/Al to increase P adsorption sites, and can also inhibit P adsorption by competition with adsorption sites.

  11. 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.

  12. 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

  13. QTL Mapping Based on Different Genetic Systems for Essential Amino Acid Contents in Cottonseeds in Different Environments

    PubMed Central

    Chen, Jinhong; Li, Jinrong; Huang, Zhuangrong; He, Qiuling; Zhu, Shuijin; Shi, Chunhai

    2013-01-01

    Cottonseeds are rich in various essential amino acids. However, the inheritance of them at molecular level are still not defined across various genetic systems. In the present study, using a newly developed mapping model that can analyze the embryo and maternal main effects as well as QTL × environment interaction effects on quantitative quality trait loci (QTLs) in cottonseeds, a study on QTL located in the tetraploid embryo and tetraploid maternal plant genomes for essential amino acid contents in cottonseeds under different environments was carried out, using the immortal F2 (IF2) populations from a set of 188 recombinant inbred lines derived from an intraspecific hybrid cross of two upland cotton germplasms HS46 and MARKCBUCAG8US-1-88 as experimental materials. The results showed a total of 35 QTLs associated with these quality traits in cottonseeds. Nineteen QTLs were subsequently mapped on chromosome 5, 6 and 8 in sub-A genome and chromosome 15, 18, 22 and 23 in sub-D genome. Eighteen QTLs were also found having QTL × environment (QE) interaction effects. The genetic main effects from QTLs located on chromosomes in the embryo and maternal plant genomes and their QE effects in different environments were all important for these essential amino acids in cottonseeds. The results suggested that the influence of environmental factors on the expression of some QTLs located in different genetic systems should be considered when improving for these amino acids. This study can serve as the foundation for the improvement of these essential amino acids in cottonseeds. PMID:23555562

  14. 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

  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.

  16. 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

  17. A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli.

    PubMed

    Knuuti, Juho; Belevich, Galina; Sharma, Vivek; Bloch, Dmitry A; Verkhovskaya, Marina

    2013-12-01

    Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2 ). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD(+) -bound enzyme as one feature of the substrate-binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.

  18. The development of essential fatty acid deficiency in healthy men fed fat-free diets intravenously and orally.

    PubMed

    Wene, J D; Connor, W E; DenBesten, L

    1975-07-01

    The hypothesis that clinical and biochemical essential fatty acid deficiency (EFA) might occur from the feeding of eucaloric, fat-free diets was tested in two experiments in healthy men. In Study I, eight men were given fat-free, eucaloric diets containing 80% of calories as glucose and 20% as amino acid hydrolysates by a constant drip over a 24-h period. The diets were fed in succession for periods of 2 wk each, either through a superior vena cava catheter or via a nasogastric tube. EFA deficiency was detected by decreases in linoleic acid and by the appearance of 5, 8, 11-eicosatrienoic acid in lipid fractions of plasma. Linoleic acid decreased significantly during 2 wk of the fat-free diet given intravenously from 48.8 to 9.8% (percent of total fatty acids) in cholesterol esters, from 21.2 to 3.2% in phospholipids, from 9.6 to 2.0% in free fatty acids, and from 14.1 to 2.6% in triglycerides. Eicosatrienoic acid, normally undetectable, appeared 0.6% in cholesterol esters, 2.5% in phospholipids, 0.2% in free fatty acids, and 2.3% in triglycerides. EFA deficiency occurred similarly during the nasogastric feeding. In Study II a subject received the same diet continuously by the nasogastric route for 10 days followed by a 24-h fast. He was then given the fat-free diet intermittently in three meals per day for 3 days. Finally, he was repleted with a diet containing 2.6% linoleic acid. By the 3rd day of the continuous nasogastric feeding, linoleic acid had fallen significantly and eicosatrienoic acid had appeared in plasma lipid fractions as in Study I. These findings were accentuated by day 10. Adipose tissue fatty acid composition did not change. Free fatty acid outflow from adipose tissue was presumably suppressed during the 10 days of continuous feeding. With increased free fatty acid outflow during fasting and intermittent feeding, linoleic acid rose and eicosatrienoic acid decreased. After 13 days of repletion with dietary linoleic acid, the EFA deficiency

  19. 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

  20. Lipid content and fatty acid composition in foods commonly consumed by nursing Congolese women: incidences on their essential fatty acid intakes and breast milk fatty acids.

    PubMed

    Rocquelin, G; Tapsoba, S; Mbemba, F; Gallon, G; Picq, C

    1998-09-01

    The fat content and fatty acid (FA) composition of nearly 40 foods, currently consumed by 102 nursing Congolese mothers living in Brazzaville, were determined to assess their impact on mothers' essential fatty acid (EFA) intakes and breast milk FA. Data on mothers' milk FA and dietary habits which allowed food selection were recently published (Rocquelin et al., 1998). Most foods were locally produced. Food samples were collected at local markets, bleached if necessary to avoid microbial degradation, and stored at +4 degrees C or -20 degrees C. They were lyophilized upon their arrival in the laboratory before lipid analyses. FA composition of food lipids was determined by capillary gas chromatography. Staple diets included low-fat, high-carbohydrate foods (processed cassava roots, wheat bread) and high-polyunsaturated fatty acid (PUFA) foods: soybean oil (high in 18 : 2 n-6 and alpha-18 : 3 n-3), bushbutter (dacryodes edulis), peanuts, avocado (high in fat and 18 : 2 n-6), freshwater and salt-water fish (high in LC n-3 and/or n-6 PUFA), and leafy green vegetables (low in fat but very high in alpha-18 : 3 n-3). Their frequent consumption by nursing mothers provided enough EFA to meet requirements due to lactation. It also explains why mothers' breast milk was rich in C8-C14 saturated FA (26% of total FA) and in n-6, n-3 PUFA (respectively 15.0% and 2.4% of total FA) highly profitable for breastfed infants' development. From this point of view, dietary habits of Congolese mothers have to be sustained for they are more adequate than most Western-type diets.

  1. 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.

  2. Threonine deprivation rapidly activates the system A amino acid transporter in primary cultures of rat neurons from the essential amino acid sensor in the anterior piriform cortex.

    PubMed

    Blais, Anne; Huneau, Jean-François; Magrum, Linda J; Koehnle, Thomas J; Sharp, James W; Tomé, Daniel; Gietzen, Dorothy W

    2003-07-01

    Omnivores show recognition of essential (indispensable) amino acid deficiency by changing their feeding behavior within 20 min, yet the cellular mechanisms of amino acid sensation in eukaryotes are poorly understood. The anterior piriform cortex (APC) of the brain in rats or its analog in birds likely houses the in vivo amino acid chemosensor. Because amino acid transporters adapt rapidly to essential amino acid deficiency in several cell models, we hypothesized that activation of electrogenic amino acid transport in APC neurons might contribute to the function of the amino acid sensor. We evaluated transport systems in primary cultures of neurons from the APC, hippocampus and cerebellum, or glia, incubated in complete or threonine-devoid (deficient) medium. After 10 min in deficient medium, uptake of threonine or a system A-selective substrate, methyl amino-isobutyric acid, was increased 60% in APC neurons only (P < 0.05). These results demonstrated upregulation of system A, an electrogenic amino acid-sodium symporter. This depletion-induced activation required sodium, intact intracellular trafficking, and phosphorylation of signal transduction-related kinases. Efflux studies showed that other transporter types were functional in the APC; they appeared to be altered dynamically in threonine-deficient cells in response to rapid increases in system A activity. The present data provided support for the chemical sensitivity of the APC and its role as the brain area housing the indispensable amino acid chemosensor. They also showed a region-specific, phosphorylation-dependent activation of the system A transporter in the brain in response to threonine deficiency.

  3. 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

  4. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  5. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  6. 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

  7. The isotopic composition of zinc, palladium, silver, cadmium, tin, and tellurium in acid-etched residues of the Allende meteorite

    SciTech Connect

    Loss, R.D.; Rosman, K.J.R.; De Laeter, J.R. )

    1990-12-01

    The isotopic and elemental abundances of Zn, Pd, Ag, Cd, Sn, and Te have been measured in three acid-resistant residues extracted from the Allende meteorite. High-efficiency, low-contamination ion-exchange procedures were developed to separate and purify the nanogram amounts of these elements present. Elemental-abundance determinations performed by Mass Spectrometric Isotope Dilution agree with previously published work for similarly derived residues. No isotope anomalies similar to those found for Xe (Xe-HL) in these samples were detected for any of these elements, which is consistent with the residues not being derived directly from the Xe-HL carriers. The lack of major Te-isotope anomalies does not support earlier reports of {sup 126}Te and {sup 130}Te excesses which were measured by neutron activation in similar samples. Small excesses were detected in the minor isotopes of Sn and Te, but these may be due to measurement problems associated with the small ion currents obtained for these samples. Two of the residue solutions contain Cd with up to several percent excesses for {sup 106}Cd and {sup 108}Cd. Interpretations of these results are limited by the unknown nature of the carrier minerals in the residues but may indicate the presence of a p-process component in Allende residues.

  8. A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

    PubMed

    Hansson, Karin; Thämlitz, Ann-Marie; Furie, Bruce; Furie, Barbara C; Stenflo, Johan

    2006-10-24

    Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

  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.

  10. Parenteral safflower oil emulsion (Liposyn 10%): safety and effectiveness in treating or preventing essential fatty acid deficiency in surgical patients.

    PubMed Central

    Bivins, B A; Rapp, R P; Record, K; Meng, H C; Griffen, W O

    1980-01-01

    The safety and effectiveness of a 10% safflower oil emulsion in treating or preventing essential fatty acid deficiency was tested in a prospective study of 15 surgical patients requiring total parenteral nutrition for two to four weeks. Three dosage regimens were evaluated including: Group I: 4% of calories as linoleate daily (five patients), Group II: 4% of calories as linoleate every other day (two patients), and Group III: 8% of calories every other day (eight patients). Patients were monitored for laboratory changes from baseline specifically in those areas where previous fat emulsions have caused serious deviations. No significant changes were noted in hematologic parameters, coagulation studies, cholesterol and triglyceride serum levels. Although there were sporadic mild deviations in liver function changes in several patients, no clinically significant adverse effects could be directly attributed to infusion of the fat emulsion. Three patients had baseline triene/tetraene ratios of 0.4 or greater, indicative of essential fatty/acid deficiency, and these ratios dropped to less than 0.4 within eight days of beginning therapy with the parenteral fat emulsion. The remaining 12 patients maintained a normal triene/tetraene ratio of less than 0.4 throughout the 28 day study period. All three dosage regimens were considered effective for treatment and prevention of essential fatty acid deficiency. Images Fig. 1. Fig. 2. Fig. 3. PMID:6767452

  11. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  12. 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

  13. 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.

  14. 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.

  15. A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL185.

    PubMed

    Shin, Hyun-Dong; McClendon, Shara; Le, Tien; Taylor, Frank; Chen, Rachel Ruizhen

    2006-12-20

    An economic ferulic acid recovery from biomass via biological methods is of interest for a number of reasons. Ferulic acid is a precursor to vanillin synthesis. It is also a known antioxidant with potential food and medical applications. Despite its universal presence in all plant cell wall material, the complex structure of the plant cell wall makes ferulic acid recovery from biomass a challenging bioprocess. Previously, without pretreatment, very low (3-13%) recovery of ferulic acid from corn residues was achieved. We report here the discovery of a filamentous fungus Neosartorya spinosa NRRL185 capable of producing a full complement of enzymes to release ferulic acid and the development of an enzymatic process for a complete recovery of ferulic acid from corn bran and corn fibers. A partial characterization of the extracellular proteome of the microbe revealed the presence of at least seven cellulases and hemicellulases activities, including multiple iso-forms of xylanase and ferulic acid esterase. The recovered ferulic acid was bio-converted to vanillin, demonstrating its potential application in natural vanillin synthesis. The enzymatic ferulic acid recovery accompanied a significant release of reducing sugars (76-100%), suggesting much broader applications of the enzymes and enzyme mixtures from this organism.

  16. Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

    PubMed

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Dietary influences of evening primrose and fish oil on the skin of essential fatty acid-deficient guinea pigs.

    PubMed

    Chapkin, R S; Ziboh, V A; McCullough, J L

    1987-08-01

    There have been reports that certain dietary lipids are capable of regulating cellular inflammation and hyperproliferation. To investigate further the role of dietary manipulation involving gamma-linolenic acid (18:3n-6) and eicosapentaenoic acid (20:5n-3) on hyperproliferative cellular components, the effects of orally administered primrose oil (containing 18:3n-6) and menhaden fish oil (containing 20:5n-3) were tested in a cutaneous system using the essential fatty acid (EFA)-deficient guinea pig fed a hydrogenated coconut oil (HCO) diet. The effects of the dietary crossover regimen were determined on epidermal 1) morphology, 2) DNA synthesis, 3) delta 6- and delta 5-desaturase activities and 4) fatty acid composition of skin and liver lipids. Our results demonstrated that dietary fish oil lacked the capacity to reverse the signs of epidermal hyperproliferation, acanthosis and hypergranulosis that are characteristic of EFA deficiency. In contrast, primrose oil feeding reversed the histological and biochemical signs of hyperproliferation. These results suggest that dietary fish oil, which contains largely the 20:5n-3 fatty acid, lacks EFA-functional properties in the skin. In addition, substitution of HCO with primrose or fish oil after 6 wk revealed incorporation of 18:3n-6 and 20:5n-3 into epidermal lipids, respectively. The significance of these altered epidermal fatty acid profiles is discussed.

  2. Determination of essential fatty acid composition among mutant lines of Canola (Brassica napus), through high pressure liquid chromatography.

    PubMed

    Raza, Ghulam; Siddique, Aquil; Khan, Imtiaz Ahmad; Ashraf, Muhammed Yasin; Khatri, Abdullah

    2009-12-01

    The present study aimed to quantify the methyl esters of lenoleic acid (LA), gamma-lenolenic acid (LNA) and oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of B. napus cv. 'Rainbow' (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. 'Westar' (P) at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.1 to 66.3%; LA was second (15.3-41.6%) and LNA was third (18.1-28.9%). Mutant ROO-125/14 showed higher OL contents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials (NUVYT) as a new variety based on high oil quality.

  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. Lipoprotein(a), essential fatty acid status and lipoprotein lipids in female Australian vegetarians.

    PubMed

    Li, D; Ball, M; Bartlett, M; Sinclair, A

    1999-08-01

    In the present study we investigated serum lipoprotein(a) [Lp(a)] levels, plasma lipids, the serum phospholipid polyunsaturated fatty acid profile and correlates of serum Lp(a) in healthy free-living female vegetarians (n=50) and omnivores (n=24) to assess differences which may have implications for cardiovascular risk. Dietary saturated fat and total plasma cholesterol were significantly lower in the vegetarians compared with omnivores. The mean serum Lp(a) concentration was lower in the vegetarians (171 mg/l) than in the omnivores (247 mg/l). The serum Lp(a) concentration was significantly negatively correlated with carbohydrate intake (as % of energy), and positively correlated with plasma total cholesterol. Compared with the omnivores, the vegetarians had significantly lower concentrations of 20:3,n-6, 20:4,n-6, 22:5,n-6, 20:5,n-3, 22:6,n-3 and total n-6 and n-3 polyunsaturated fatty acids, and a lower n-3/n-6 polyunsaturated fatty acid ratio, in serum phospholipids. Lower concentrations of plasma total cholesterol, serum phospholipid total fatty acids, total saturated fatty acids and arachidonic acid, and a tendency towards a lower serum Lp(a) concentration, in vegetarians may have beneficial effects on cardiovascular disease risk. However, the decreased concentration of serum phospholipid n-3 polyunsaturated fatty acids may potentially promote thrombotic risk. Based on the present data, it would seem appropriate for omnivores to reduce their dietary intake of total fat and saturated fat in order to decrease their plasma cholesterol, and vegetarians should perhaps increase their dietary intake of n-3 polyunsaturated fatty acids, and thus improve the balance of n-3/n-6, in order to reduce any thrombotic tendency that might increase their generally low risk of cardiovascular disease.

  5. Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function.

    PubMed

    Qin, Lei; Tam, Shui-Pang; Deeley, Roger G

    2012-07-01

    Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

  6. 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

  7. 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.

  8. 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

  9. Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet.

    PubMed

    Ghioni, C; Bell, J G; Bell, M V; Sargent, J R

    1997-06-01

    Rainbow trout (Oncorhynchus mykiss) were fed either a control diet containing fish oil or an essential fatty acid (EFA) deficient diet containing only hydrogenated coconut oil and palmitic acid as lipid source (93.4% saturated fatty acids) for 14 weeks and the fatty acid compositions of individual phospholipid classes from skin and opercular membrane (OM) determined. The permeability of skin and OM to water and the production of eicosanoids in skin and gills challenged with the Ca2+ ionophore A23187 were also measured. Phospholipid (PL) fatty acid compositions were substantially modified in EFA-deficient fish, with increased saturated fatty acids and decreased polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) was largely retained. The onset of EFA deficiency was shown by the appearance of n-9 PUFA, particularly 20:3n-9. The main effects of EFA deficiency on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were to increase saturated fatty acids and monoenes, especially 16:1 and 18:1, and to decrease EPA and DHA. The content of DHA in phosphatidylserine (PS) was high in control animals (40% in skin and 35% in opercular membrane) and was mostly retained in EFA deficient animals. Arachidonic acid (AA) was the most abundant PUFA esterified to phosphatidylinositol (PI) and was significantly reduced in EFA deficient animals (from 31% to 13% in skin), where a large amount of 20:3n-9 (9% in skin) was also present. Influxes and effluxes of water through skin and opercular membrane were measured in vitro. No differences were detected between rainbow trout fed the control or the EFA deficient diet. 12-Hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHE) could not be detected in skin from control or EFA deficient fish. There was no difference between control and EFA deficient trout in the levels of leukotriene C

  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. 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.

  12. Influence of certain essential oils on carcinogen-metabolizing enzymes and acid-soluble sulfhydryls in mouse liver.

    PubMed

    Banerjee, S; Sharma, R; Kale, R K; Rao, A R

    1994-01-01

    The influence of essential oils from naturally occurring plant dietary items such as cardamom, celery seed, cumin seed, coriander, ginger, nutmeg, and zanthoxylum on the activities of hepatic carcinogen-metabolizing enzymes (cytochrome P450, aryl hydrocarbon hydroxylase, and glutathione S-transferase) and acid-soluble sulfhydryl level was investigated in Swiss albino mice. Each oil was fed by gavage at 10 microliters/day for 14 days, and then the animals were sacrificed and their hepatic enzyme activities and sulfhydryl levels were evaluated. Only nutmeg and zanthoxylum oils induced cytochrome P450 level significantly (p < 0.05), whereas cardamom oil caused a significant reduction in its activity (p < 0.05). Furthermore, aryl hydrocarbon hydroxylase activity was significantly elevated only by treatment with ginger oil (p < 0.01), whereas nutmeg oil caused a significant reduction in its activity (p < 0.01). The remaining oils did not significantly alter the level of cytochrome P450 and aryl hydrocarbon hydroxylase activity. Glutathione S-transferase activity was significantly elevated in all experimental groups (p < 0.1-p < 0.001) compared with controls. The acid-soluble sulfhydryl was significantly elevated only by the essential oils of cardamom (p < 0.05), nutmeg (p < 0.05), and zanthoxylum (p < 0.01). Our observations suggest that intake of essential oils affects the host enzymes associated with activation and detoxication of xenobiotic compounds, including chemical carcinogens and mutagens. PMID:8072879

  13. Antimicrobial efficacy of emulsified essential oil components against weak acid-adapted spoilage yeasts in clear and cloudy apple juice.

    PubMed

    Loeffler, Myriam; Beiser, Sophia; Suriyarak, Sarisa; Gibis, Monika; Weiss, Jochen

    2014-08-01

    The antimicrobial activity of oil-in-water emulsions containing dual combinations of the essential oil components cinnamaldehyde, perillaldehyde, and citral was examined against two acid-resistant yeast strains (Zygosaccharomyces bailii) in beverage systems composed of diluted clear or cloudy apple juice and in a Sabouraud dextrose broth model. Antimicrobial properties of an encapsulated oil-in-water emulsion and of essential oil components dissolved in 10% dimethyl sulfoxide were compared using plate counts and turbidity measurements. Growth curves were modulated to qualitatively assess differences in antimicrobial efficacy. The impact of the presence of a beverage emulsion without essential oils (unloaded; 5% oil and 1% modified starch, pH 3.0) on the antimicrobial efficacy also was investigated. Dual combinations of essential oil components were sufficient to completely inhibit and/or kill yeast cells in diluted apple juice and Sabouraud dextrose broth systems at very low concentrations (100 to 200 μg/ml). However, the combination of perillaldehyde and citral had the weakest antimicrobial effect; a concentration of 400 μg/ml was necessary to prevent yeast growth in beverages, and up to 800 μg/ml was required in systems to which an unloaded emulsion had been added. The antimicrobial activity of essential oil components did not differ in diluted clear and cloudy apple juices and was not affected by being added in emulsified form or dissolved in dimethyl sulfoxide as long as there was no unloaded emulsion also present. These results indicate that formulations of essential oil combinations encapsulated together in emulsions are highly effective for inhibiting and/or killing microorganisms in real beverage systems.

  14. Increasing intake of essential fatty acids from milk replacer benefits performance, immune responses, and health of preweaned Holstein calves.

    PubMed

    Garcia, M; Shin, J H; Schlaefli, A; Greco, L F; Maunsell, F P; Thatcher, W W; Santos, J E P; Staples, C R

    2015-01-01

    The objective was to evaluate the effect of feeding increasing amounts of essential fatty acids (FA) in milk replacer (MR) during the first 60 d of life on growth, health, and immunity of Holstein calves. Calves were born from dams fed low concentrations of total and essential FA during the lasT2 mo of pregnancy. Newborn calves were blocked by sex and parity of the dam and assigned randomly to receive 1 of 4 MR treatments (T). Hydrogenated coconut oil and soybean oil were mixed with emulsifier and commercial MR powder to prepare the following 4 MR containing 0.119 and 0.007 (T1), 0.187 and 0.017 (T2), 0.321 and 0.036 (T3), and 0.593 and 0.076 (T4) g of intake per kg of metabolic body weight (BW(0.75)) of linoleic acid and α-linolenic acid, respectively. At 30 d of life, concentrations of essential FA (linoleic acid and α-linolenic acid) in liver increased, whereas concentrations of C12:0, C14:0, C16:0, and C20:3n-9 decreased linearly with increasing intake of essential FA. Body weight gain and feed efficiency were optimized when male calves consumed T2, whereas gain by female calves tended to increase linearly with increasing intake of essential FA during the first 30 d of age. However, these responses to treatment were not maintained after initiation of concentrate feeding at 31 d of life. Over the 60-d preweaning period, wither and hip heights were improved in both sexes as intake of essential FA increased up to T3. Some measures of health and immunity were affected by replacing some coconut oil with soybean oil. Severity of diarrhea tended to decrease linearly; plasma concentrations of haptoglobin during diarrhea were lower in T2, T3, and T4; phagocytosis by blood neutrophils tended to peak for calves fed T2; in vitro proliferation of stimulated blood lymphocytes was greater for calves fed T2; in vitro stimulated blood cells produced more IFN-γ (up to T3 for males and T2 for females), concentrations of serum IgG against ovalbumin injections were increased in

  15. Increasing intake of essential fatty acids from milk replacer benefits performance, immune responses, and health of preweaned Holstein calves.

    PubMed

    Garcia, M; Shin, J H; Schlaefli, A; Greco, L F; Maunsell, F P; Thatcher, W W; Santos, J E P; Staples, C R

    2015-01-01

    The objective was to evaluate the effect of feeding increasing amounts of essential fatty acids (FA) in milk replacer (MR) during the first 60 d of life on growth, health, and immunity of Holstein calves. Calves were born from dams fed low concentrations of total and essential FA during the lasT2 mo of pregnancy. Newborn calves were blocked by sex and parity of the dam and assigned randomly to receive 1 of 4 MR treatments (T). Hydrogenated coconut oil and soybean oil were mixed with emulsifier and commercial MR powder to prepare the following 4 MR containing 0.119 and 0.007 (T1), 0.187 and 0.017 (T2), 0.321 and 0.036 (T3), and 0.593 and 0.076 (T4) g of intake per kg of metabolic body weight (BW(0.75)) of linoleic acid and α-linolenic acid, respectively. At 30 d of life, concentrations of essential FA (linoleic acid and α-linolenic acid) in liver increased, whereas concentrations of C12:0, C14:0, C16:0, and C20:3n-9 decreased linearly with increasing intake of essential FA. Body weight gain and feed efficiency were optimized when male calves consumed T2, whereas gain by female calves tended to increase linearly with increasing intake of essential FA during the first 30 d of age. However, these responses to treatment were not maintained after initiation of concentrate feeding at 31 d of life. Over the 60-d preweaning period, wither and hip heights were improved in both sexes as intake of essential FA increased up to T3. Some measures of health and immunity were affected by replacing some coconut oil with soybean oil. Severity of diarrhea tended to decrease linearly; plasma concentrations of haptoglobin during diarrhea were lower in T2, T3, and T4; phagocytosis by blood neutrophils tended to peak for calves fed T2; in vitro proliferation of stimulated blood lymphocytes was greater for calves fed T2; in vitro stimulated blood cells produced more IFN-γ (up to T3 for males and T2 for females), concentrations of serum IgG against ovalbumin injections were increased in

  16. 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

  17. 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

  18. Residual Chemoresponsiveness to Acids in the Superior Laryngeal Nerve in “Taste-Blind” (P2X2/P2X3 Double-KO) Mice

    PubMed Central

    Ohkuri, Tadahiro; Horio, Nao; Stratford, Jennifer M.; Finger, Thomas E.; Ninomiya, Yuzo

    2012-01-01

    Mice lacking both the P2X2 and the P2X3 purinergic receptors (P2X-dblKO) exhibit loss of responses to all taste qualities in the taste nerves innervating the tongue. Similarly, these mice exhibit a near total loss of taste-related behaviors in brief access tests except for a near-normal avoidance of acidic stimuli. This persistent avoidance of acids despite the loss of gustatory neural responses to sour was postulated to be due to continued responsiveness of the superior laryngeal (SL) nerve. However, chemoresponses of the larynx are attributable both to taste buds and to free nerve endings. In order to test whether the SL nerve of P2X-dblKO mice remains responsive to acids but not to other tastants, we recorded responses from the SL nerve in wild-type (WT) and P2X-dblKO mice. WT mice showed substantial SL responses to monosodium glutamate, sucrose, urea, and denatonium—all of which were essentially absent in P2X-dblKO animals. In contrast, the SL nerve of P2X-dblKO mice exhibited near-normal responses to citric acid (50 mM) although responsiveness of both the chorda tympani and the glossopharyngeal nerves to this stimulus were absent or greatly reduced. These results are consistent with the hypothesis that the residual avoidance of acidic solutions by P2X-dblKO mice may be attributable to the direct chemosensitivity of nerve fibers innervating the laryngeal epithelium and not to taste. PMID:22362867

  19. Essential role for retinoic acid in the promotion of CD4+ T cell effector responses via retinoic acid receptor alpha

    PubMed Central

    Hall, J.A.; Cannons, J.L.; Grainger, J.R.; Santos, L.M. Dos; Hand, T.W.; Naik, S.; Wohlfert, E.A.; Chou, D.B.; Oldenhove, G.; Robinson, M.; Grigg, M.E.; Kastenmayer, R.; Schwartzberg, P.L.; Belkaid, Y.

    2012-01-01

    SUMMARY Vitamin A and its metabolite, retinoic acid (RA), have recently been implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we show that RA is also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) is the critical mediator of these effects. Strikingly, antagonism of RAR signaling and deficiency in RARα(Rara−/−) results in a cell autonomous CD4+ T cell activation defect. Altogether, these findings reveal a fundamental role for the RA/RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses. PMID:21419664

  20. Abnormal essential fatty acid composition of tissue lipids in genetically diabetic mice is partially corrected by dietary linoleic and gamma-linolenic acids.

    PubMed

    Cunnane, S C; Manku, M S; Horrobin, D F

    1985-05-01

    Genetically diabetic mice (db/db) and their non-diabetic litter-mates were maintained for 15 weeks on diets supplemented with safflower oil or evening primrose (Oenothera bienis) oil, both essential fatty acid (EFA)-rich sources, or hydrogenated coconut oil (devoid of EFA). Plasma glucose was higher in the diabetic mice supplemented with the oils than in the unsupplemented diabetic mice. In the oil-supplemented non-diabetic mice, plasma glucose did not differ compared with the unsupplemented non-diabetic mice. The proportional content of arachidonic acid in the phospholipids of the pancreas was significantly decreased in diabetic mice, an effect which was completely prevented by supplementation with safflower or evening primrose oil but not hydrogenated coconut oil. In the liver phospholipids of the diabetic mice, dihomo-gamma-linolenic acid was proportionally increased, an effect reduced by supplementation with safflower oil but not evening primrose or hydrogenated coconut oils. In the liver triglycerides of the diabetic mice, gamma-linolenic acid, dihomo-gamma-linolenic acid and arachidonic acid were all proportionally decreased, effects which were also prevented by safflower or evening primrose oil but not hydrogenated coconut oil. Alopecia and dry scaly skin were prominent in the diabetic mice but less extensive in the diabetic mice supplemented with EFA.

  1. Urea, Glycolic Acid, and Glycerol in an Organic Residue Produced by Ultraviolet Irradiation of Interstellar/Pre-Cometary Ice Analogs

    NASA Astrophysics Data System (ADS)

    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 CH3OH:NH3â = 1:1 ice mixture was UV irradiated at ˜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.

  2. 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.

  3. 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

  4. [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.

  5. 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

  6. Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase.

    PubMed

    Alexeyev, Mikhail F; Winkler, Herbert H

    2002-09-20

    The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches. PMID:12225862

  7. 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.

  8. 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.

  9. 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.

  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. 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

  12. Dynamic HypA zinc site is essential for acid viability and proper urease maturation in Helicobacter pylori

    PubMed Central

    Johnson, Ryan C.; Hu, Heidi Q.; Merrell, D. Scott; Maroney, Michael J.

    2015-01-01

    Helicobacter pylori requires urease activity in order to survive in the acid environment of the human stomach. Urease is regulated in part by nickelation, a process that requires the HypA protein, which is a putative nickel metallochaperone that is generally associated with hydrogenase maturation. However, in H. pylori, HypA plays a dual role. In addition to an N-terminal nickel binding site, HypA proteins also contain a structural zinc site that is coordinated by two rigorously conserved CXXC sequences, which in H. pylori are flanked by His residues. These structural Zn sites are known to be dynamic, converting from Zn(Cys)4 centers at pH 7.2 to Zn(Cys)2(His)2 centers at pH 6.3 in the presence of Ni(II) ions. In this study, mutant strains of H. pylori that express zinc site variants of the HypA protein are used to show that the structural changes in the zinc site are important for the acid viability of the bacterium, and that a reduction in acid viability in these variants can be traced in large measure to deficient urease activity. This in turn leads to a model that connects the Zn(Cys)4 coordination to urease maturation. PMID:25608738

  13. Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

    PubMed Central

    Purasiri, P; Mckechnie, A; Heys, S D; Eremin, O

    1997-01-01

    Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities. PMID:9415022

  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. 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.

  16. Identification and quantitative determination of eudesmane-type acids from the essential oil of Dittrichia viscosa sp. viscosa using 13C-NMR spectroscopy.

    PubMed

    Blanc, Marie-Cécile; Bradesi, Pascale; Casanova, Joseph

    2005-01-01

    A procedure that allows the identification and quantitative determination of eudesmane-type acids in the acidic part of the essential oil of Dittrichia viscosa sp. viscosa is described. The method involves the computer-aided analysis of the 13C-NMR spectrum of the mixture without the requirement of previous separation or derivatisation. The quantitative procedure was verified with costic acid standard and applied to three other acids which possess the same eudesmane framework.

  17. A Peculiar Formula of Essential Amino Acids Prevents Rosuvastatin Myopathy in Mice

    PubMed Central

    D'Antona, Giuseppe; Tedesco, Laura; Ruocco, Chiara; Corsetti, Giovanni; Ragni, Maurizio; Fossati, Andrea; Saba, Elisa; Fenaroli, Francesca; Montinaro, Mery; Carruba, Michele O.; Valerio, Alessandra

    2016-01-01

    Abstract Aims: Myopathy, characterized by mitochondrial oxidative stress, occurs in ∼10% of statin-treated patients, and a major risk exists with potent statins such as rosuvastatin (Rvs). We sought to determine whether a peculiar branched-chain amino acid-enriched mixture (BCAAem), found to improve mitochondrial function and reduce oxidative stress in muscle of middle-aged mice, was able to prevent Rvs myopathy. Results: Dietary supplementation of BCAAem was able to prevent the structural and functional alterations of muscle induced by Rvs in young mice. Rvs-increased plasma 3-methylhistidine (a marker of muscular protein degradation) was prevented by BCAAem. This was obtained without changes of Rvs ability to reduce cholesterol and triglyceride levels in blood. Rather, BCAAem promotes de novo protein synthesis and reduces proteolysis in cultured myotubes. Morphological alterations of C2C12 cells induced by statin were counteracted by amino acids, as were the Rvs-increased atrogin-1 mRNA and protein levels. Moreover, BCAAem maintained mitochondrial mass and density and citrate synthase activity in skeletal muscle of Rvs-treated mice beside oxygen consumption and ATP levels in C2C12 cells exposed to statin. Notably, BCAAem assisted Rvs to reduce oxidative stress and to increase the anti-reactive oxygen species (ROS) defense system in skeletal muscle. Innovation and Conclusions: The complex interplay between proteostasis and antioxidant properties may underlie the mechanism by which a specific amino acid formula preserves mitochondrial efficiency and muscle health in Rvs-treated mice. Strategies aimed at promoting protein balance and controlling mitochondrial ROS level may be used as therapeutics for the treatment of muscular diseases involving mitochondrial dysfunction, such as statin myopathy. Antioxid. Redox Signal. 25, 595–608. PMID:27245589

  18. 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

  19. A facile route to preparation of high purity nanoporous silica from acid-leached residue of serpentine.

    PubMed

    Bai, Penn; Sharratt, Paul; Yeo, Tze Yuen; Bu, Jie

    2014-09-01

    As the current cost of mineral carbonation is too high for an economically viable industrial process, it is desirable to produce value-added products from CO2 mineralization process. In this work, a facile and cost-effective process was developed for the production of high purity SiO2 from acid-leached serpentine residue. The Si extraction rate is fast even under ambient conditions due to the highly defective structure of the residue. The reaction kinetics were studied and it was found that the Si extraction rate was under a combination of chemical reaction control and film diffusion control. The SiO2 sample prepared has high purity with a nanoporous structure, which renders it a potential candidate for applications such as an adsorbent and a catalyst support.

  20. 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.

  1. Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue.

    PubMed

    Onishi, H; Maita, T; Miyanishi, T; Watanabe, S; Matsuda, G

    1986-12-01

    A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

  2. Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues.

    PubMed

    Roy, Rituparna S; Gopi, Hosahudya N; Raghothama, S; Gilardi, Richard D; Karle, Isabella L; Balaram, Padmanabhan

    2005-01-01

    The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also

  3. Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

    PubMed Central

    2010-01-01

    Background Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins. Results We identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2. Conclusions Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches. PMID:20840750

  4. Type II fatty acid synthesis is essential only for malaria parasite late liver stage development

    PubMed Central

    Vaughan, Ashley M; O'Neill, Matthew T; Tarun, Alice S; Camargo, Nelly; Phuong, Thuan M; Aly, Ahmed S I; Cowman, Alan F; Kappe, Stefan H I

    2009-01-01

    Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood. PMID:19068099

  5. 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

  6. The transcriptional coactivator PGC-1alpha is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis.

    PubMed

    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

    2008-07-01

    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-gamma coactivator (PGC)-1alpha is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1alpha (PGC-1alpha(-/-) mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1alpha is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1alpha(-/-) mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1alpha(-/-) mice compared with PGC-1alpha(+/+) controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1alpha(-/-) mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1alpha(-/-) fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1alpha(-/-) mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. (13)C NMR revealed that hearts from PGC-1alpha(-/-) mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during beta-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1alpha(-/-) mice relative to PGC-1alpha(+/+) following a fast. Overall, these results demonstrate that PGC-1alpha is essential for the maintenance of maximal, efficient cardiac

  7. ASCORBIC ACID REDUCTION ON RESIDUAL ACTIVE CHLORINE IN POTABLE WATER PRIOR TO HALOCARBOXYLATE DETERMINATION

    EPA Science Inventory

    In studies on the formation of disinfection byproducts (DBPs), it is necessary to scavenge residual active (odxidizing) chlorine in order to fix the chlorination byproducts (such as haloethanoates) at a point in time . Such research projects often have distinct needs from requi...

  8. ASCORBIC ACID REDUCTION OF RESIDUAL ACTIVE CHLORINE IN POTABLE WATER PRIOR TO HALOCARBOXYLATE DETERMINATION

    EPA Science Inventory

    In studies on the formation of disinfection byproducts (DBPs), it is necessary to scavenge residual active (oxidizing) chlorine in order to fix the chlorination byproducts (such as haloethanoates) at a point in time. Thus, methods designed for compliance monitoring are not alway...

  9. 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

  10. 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

    Ohara, Kazuaki; Fukuda, Takafumi; Okada, Hiroyuki; Kitao, Sayoko; Ishida, Yuko; Kato, Kyoko; Takahashi, Chika; Katayama, Mikio; Uchida, Kunitoshi; Tominaga, Makoto

    2015-01-30

    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 Ca(2+) 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

  11. 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

  12. 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.

  13. KV4.3 expression and gating: S2 and S3 acidic and S4 innermost basic residues.

    PubMed

    Skerritt, Matthew R; Campbell, Donald L

    2009-11-01

    Effects of neutralizing individual negatively charged (acidic [E,D]) and innermost positively charged (basic [K,R]) residues in transmembrane segments S2 (D230Q, E240Q), S3 (D263Q) and S4 (K299A/Q, R302A/Q) of the K(V)4.3 putative voltage sensing domain (VSD) were determined. S2 D230Q generated large macroscopic currents, depolarized steady-state activation ("a(4)") and isochronal (1 sec) inactivation ("i") relationships, and significantly accelerated kinetics of deactivation and recovery (from both macroscopic and closed state inactivation [CSI]). D230Q thus stabilized non-inactivated closed states. These effects were attributable to structural perturbations, and indicated D230 is not primarily involved in voltage sensing. Both S2 E240Q and S3 D263Q failed to generate measurable ionic currents, suggesting deletion of negative charges at these putatively more intracellular acidic positions were functionally "lethal" to macroscopic K(V)4.3 function. S4 innermost positive charge deletion mutants K299A/Q and R032A/Q generated functional currents with reduced peak amplitudes. While reduced K299A/Q and R302A/Q currents prevented accurate determination of "a(4)" and estimates of potential electrostatic perturbations, both sets of mutants: (i) depolarized potentials at which currents could be macroscopically detected, consistent with stabilization of closed states (structural perturbations); and (ii) accelerated macroscopic recovery. These results provide further evidence that: (i) basic residues in S4 are involved not only in regulating K(V)4.3 activation and deactivation, but also CSI and recovery; and (ii) suggest putative electrostatic interactions between acidic S2/S3 and basic S4 residues may be different in K(V)4.3 from those proposed to exist in Shaker. Functional implications are discussed.

  14. Intake of essential fatty acids by growing-finishing pigs kept on smallholdings in central Vietnam.

    PubMed

    Nguyen, L Q; Everts, H; Beynen, A C

    2005-01-01

    The intake of linoleic acid (LA) and alpha-linolenic acid (ALA) in relation to average daily weight gain (ADG) was studied in growing-finishing pigs kept on smallholdings in Central Vietnam. Groups of three piglets each were assigned randomly to 12 farms, where they were fed on local feedstuffs according to the farmer's choice but were given a restricted amount of dry matter according to a preset feeding regimen. On arrival at the farms, the pigs weighed 10.4 +/- 0.9 kg (mean +/- SD, n = 36) and at 130 days of age they weighed 45.4 +/- 9.6 kg. Dietary LA concentration ranged from 1.34 to 2.41 g/MJ metabolizable energy (ME) and ALA from 0.06 to 0.33 g/MJ ME. On a farm level, dietary LA and ALA concentrations were significantly correlated with their concentrations in adipose tissue, both correlation coefficients being 0.63. Dietary protein concentration and protein:energy ratio were significantly correlated with ADG, the correlation coefficients being 0.68 and 0.64. For individual piglets there were significant correlations between either LA or ALA in adipose tissue and ADG, the correlation coefficients being 0.37 and 0.45. Dietary protein concentration or protein:energy ratio was correlated with the dietary contents of LA and ALA. It is uncertain whether LA and ALA intake had a causal relationship with ADG. Since dietary LA levels were above the LA requirement, LA intake may not have limited growth. It is suggested tentatively that, through enhanced disease resistance, supplementation with ALA of the diets on the farms studied might have a positive influence on ADG.

  15. Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

    PubMed Central

    Kunz, Daniel A.; Chen, Jui-Lin; Pan, Guangliang

    1998-01-01

    Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min−1 ml of culture fluid−1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role. PMID:9797306

  16. 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.

  17. 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

  18. 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

  19. Absorption and metabolism of ( sup 3 H)arachidonic and ( sup 14 C)linoleic acid in essential fatty acid-deficient rats

    SciTech Connect

    Hjelte, L.; Melin, T.; Nilsson, A.; Strandvik, B. )

    1990-07-01

    ({sup 3}H)arachidonic acid (20:4) and ({sup 14}C)linoleic acid (18:2) were fed in a triolein emulsion to essential fatty acid-deficient (EFAD) rats and to age-matched controls. Tissues were analyzed for radioactivity of different lipid classes after 1, 2, and 4 h. As in earlier studies, control rats retained more ({sup 3}H)20:4 than ({sup 14}C)18:2 in all organs except adipose tissue. In EFAD rats, recovery of ({sup 14}C)18:2 was increased in small intestine, liver, heart, and kidneys. In comparison to controls, EFAD rats retained much more ({sup 14}C)18:2 in phospholipids of these organs. The increase in the incorporation of both {sup 3}H and {sup 14}C into phosphatidylethanolamine was particularly pronounced. Another striking feature was the drastic increase in the retention after 4 h of {sup 14}C in cardiolipin, which is specifically located in the inner mitochondrial membrane. In contrast, incorporation of both {sup 3}H and {sup 14}C into phosphatidylinositol was decreased or unchanged in EFAD rats. Although fecal fat excretion was increased there was no evidence for a malabsorption or an increased retention in intestinal triacyglycerol of the radioactive fatty acids in EFAD rats. The proportion of ({sup 14}C)18:2 that had been converted to ({sup 14}C)20:4 was generally low but increased significantly with time in the liver and intestine of EFAD rats.

  20. 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

  1. 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

  2. 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

  3. 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

  4. Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

    PubMed Central

    Chirgadze, Nickolay Y.; Briggs, Steven L.; McAllister, Kelly A.; Fischl, Anthony S.; Zhao, Genshi

    2000-01-01

    Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3′5′-ADP, a product of AcpS, at 2.0 and 1.9 Å resolution, respectively. The crystal structure reveals an α/β fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity. PMID:11032795

  5. 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

  6. 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

  7. Taurine, a conditionally essential amino acid, ameliorates arsenic-induced cytotoxicity in murine hepatocytes.

    PubMed

    Sinha, Mahua; Manna, Prasenjit; Sil, Parames C

    2007-12-01

    Arsenic is a potent environmental toxin. Present study has been designed to evaluate the protective role of taurine (2-aminoethanesulfonic acid) against arsenic induced cytotoxicity in murine hepatocytes. Sodium arsenite (NaAsO(2)) was chosen as the source of arsenic. Incubation of hepatocytes with the toxin (1 mM) for 2 h reduced the cell viability as well as intra-cellular antioxidant power. Increased activities of alanine transaminase (ALT) and alkaline phosphatase (ALP) due to toxin exposure confirmed membrane damage. Toxin treatment caused reduction in the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). In addition, the same treatment reduced the level of glutathione (GSH), elevated the level of oxidized glutathione (GSSG) and increased the extent of lipid peroxidation. Incubation of hepatocytes with taurine, both prior to and in combination with NaAsO(2), attenuated the extent of lipid peroxidation and enhanced the activities of enzymatic as well as non enzymatic antioxidants. Besides, taurine administration normalized the arsenic-induced enhanced levels of the marker enzymes ALT and ALP in hepatocytes. The cytoprotective activity of taurine against arsenic poisoning was found to be comparable to that of a known antioxidant, vitamin C. Combining all, the results suggest that taurine protects mouse hepatocytes against arsenic induced cytotoxicity.

  8. Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin

    PubMed Central

    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

    2016-01-01

    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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix.

    PubMed

    Cheong, Wan-Shoo; Drummer, Heidi Edelgard; Netter, Hans-Jürgen

    2009-06-01

    A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

  15. 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.

  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

    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.

  18. Effect of eutrophication on mercury, selenium, and essential fatty acids in bighead carp (Hypophthalmichthys nobilis) from reservoirs of eastern China.

    PubMed

    Razavi, N Roxanna; Arts, Michael T; Qu, Mingzhi; Jin, Binsong; Ren, Wenwei; Wang, Yuxiang; Campbell, Linda M

    2014-11-15

    Analyses of the risks and benefits of consuming fish assess the content of beneficial fatty acids found in fish relative to harmful pollutants such as methylmercury (MeHg). Quantifying the effect of eutrophication on mercury (Hg), selenium (Se) and essential fatty acids (EFAs) in fish is necessary to determine how measures of risk vary with productivity. Total Hg and MeHg, Se and fatty acids, including the EFA eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), were analyzed in Bighead Carp (Hypophthalmichthys nobilis) dorsal muscle tissue from seven subtropical reservoirs of eastern China. Individual elements and fatty acids, as well as derived measures of risk (Se:Hg and hazard quotient, HQ(EFA)) were regressed against indicators of eutrophication, including total phosphorous (TP), chlorophyll-a (chl-a) and phytoplankton species composition. We found low MeHg concentrations (range=0.018-0.13 μg/g ww) and Se concentrations (range=0.12-0.28 μg/g ww), and Se:Hg molar ratios that were well above 1.0, indicating a low risk of Hg toxicity. Bighead Carp had a high content of total polyunsaturated fatty acids (∑PUFAs=44.2-53.6%), which included both EPA (6.9-12.5%) and DHA (16.1-23.2%). However, fish had significantly lower Se:Hg molar ratios in reservoirs with high TP, and lower EPA content with increasing plankton density (i.e. higher chl-a). Phytoplankton species composition predicted Se concentrations, but not Hg concentrations or EFA content. Overall, Hg concentrations in Bighead Carp were very low relative to consumption guidelines, and Se concentrations were adequate to confer protective benefits against MeHg toxicity. Our findings suggest that changes to plankton species composition and density with eutrophication may result in fish of lower nutritional value and thus increase risks to fish consumers by changing the availability of Se and EPA relative to MeHg. PMID:25173860

  19. Prevention of diabetes in the BB rat by essential fatty acid deficiency. Relationship between physiological and biochemical changes

    PubMed Central

    1990-01-01

    Essential fatty acid (EFA) deficiency exerts a striking protective effect in several animal models of autoimmune disease. We now report that EFA deprivation prevents diabetes in the BB rat, an animal model of human insulin-dependent diabetes mellitus. In diabetes-prone (DP)-BB rats, the incidences of spontaneous diabetes and insulitis (the pathological substrate of autoimmune diabetes) were greatly reduced by EFA deficiency. This beneficial effect of the deficiency state was also seen in diabetes-resistant (DR)-BB rats that, after treatment with antibody to eliminate RT6+ T cells, would otherwise have become diabetic. The susceptibility of EFA-deprived DP-BB rats to spontaneous diabetes was restored when they were given dietary supplements of linoleate at 70 d of age (during the usual period of susceptibility), but not when they were repleted beginning at 120 d (after the peak incidence of diabetes). EFA deficiency did lead to growth retardation, but calorically restricted control rats demonstrated that the protective effect of the deficiency state was not a function of decreased weight. To examine the relationship between the biochemical changes of EFA deficiency and its physiological effects in this system, we compared the fatty acid changes that occurred in EFA-deficient animals that did and did not develop diabetes. Nondiabetic animals had significantly lower levels of (n-6) fatty acids (i.e., linoleate and arachidonate) and higher levels of oleate, an (n-9) fatty acid, than did diabetic animals. Levels of 20:3(n