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Sample records for aspartic endopeptidases

  1. Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

    Science.gov (United States)

    Rojo, Liliana; Muhlia-Almazan, Adriana; Saborowski, Reinhard; García-Carreño, Fernando

    2010-11-01

    Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding. PMID:20169386

  2. Multiple forms of endopeptidase activity from jojoba seeds.

    Science.gov (United States)

    Wolf, M J; Storey, R D

    1990-01-01

    The cotyledons of 27 day post-germination jojoba seedlings (Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their Mr and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and Mr of 58,000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and Mr of 41,000, 47,000 and 35,000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and Mr of 33,000.

  3. Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (Neprilysin)

    NARCIS (Netherlands)

    Lian, W; Wu, D; Konings, W.N; Mierau, I; Hersh, L.B

    1996-01-01

    A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was clone

  4. Induced-fit Mechanism for Prolyl Endopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Min; Chen, Changqing; Davies, David R.; Chiu, Thang K. (NIH); (LSU); (Chinese Aca. Sci.)

    2010-11-15

    Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.

  5. Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid.

    Science.gov (United States)

    Risør, Michael W; Thomsen, Line R; Sanggaard, Kristian W; Nielsen, Tania A; Thøgersen, Ida B; Lukassen, Marie V; Rossen, Litten; Garcia-Ferrer, Irene; Guevara, Tibisay; Scavenius, Carsten; Meinjohanns, Ernst; Gomis-Rüth, F Xavier; Enghild, Jan J

    2016-01-29

    Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms. PMID:26627834

  6. Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid.

    Science.gov (United States)

    Risør, Michael W; Thomsen, Line R; Sanggaard, Kristian W; Nielsen, Tania A; Thøgersen, Ida B; Lukassen, Marie V; Rossen, Litten; Garcia-Ferrer, Irene; Guevara, Tibisay; Scavenius, Carsten; Meinjohanns, Ernst; Gomis-Rüth, F Xavier; Enghild, Jan J

    2016-01-29

    Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.

  7. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    Science.gov (United States)

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  8. Changes in Endopeptidase Activity during Photosynthetic Declination in Rice Leaf

    Institute of Scientific and Technical Information of China (English)

    DENGZhi-rui; ZHANGRong-xian

    2004-01-01

    Two japonica rice varieties, Wuyujing 3 and 97-7, were used to study the changes in contents of soluble protein, free amino acids and endopeptidase activity, during photosynthetic declination. The content of soluble protein in flag leaf of cv.Wuyujing 3 was higher than that of cv. 97-7, but decreased rapidly in Wuyujing 3. Free amino acids in flag leaf and the thirteenth leaf of Wuyujing 3 started to increase 10 days before the turning point of photosynthetic declination (TPPD), while it occurred just 1-2 days before TPPD in the flag leaf and the thirteenth leaf of 97-7. During reversible phase of photosynthetic declination,endopeptidase activity remained at a low level and increased slightly only in the later part of this phase. Then it rose up rapidly at irreversible decline phase and reached a vety high level. For Wuyujing 3, the change in endopeptidase activity in the thirteenth leaf was parallel to that in flag leaf. However, for 97-7, the rapid increase of endopeptidase activity in the thirteenth leaf started later than that of flag leaf. The results implied that the rate of protein breakdown and conversion to transportable nitrogen in leaves of 97-7 was slower than that in leaves of Wuyujing 3 during photosynthetic declination and it led to relativeh" lower seed setting rate and fully filling grains rate in 97-7. This may be one of the important reasons why 97-7 could not bring the high yicld potentiality into play and the findings may be taken into consideration while breeding for high potential varieties in future.

  9. Changes in Endopeptidase Activity during Photosynthetic Declination in Rice Leaf

    Institute of Scientific and Technical Information of China (English)

    DENG Zhi-rui; ZHANG Rong-xian

    2004-01-01

    Two japonica rice varieties, Wuyujing 3 and 97-7, were used to study the changes in contents of soluble protein, free amino acids and endopeptidase activity during photosynthetic declination. The content of soluble protein in flag leaf of cv.Wuyujing 3 was higher than that of cv. 97-7, but decreased rapidly in Wuyujing 3. Free amino acids in flag leaf and the thirteenth leaf of Wuyujing 3 started to increase 10 days before the turning point of photosynthetic declination (TPPD), while it occurred just 1-2 days before TPPD in the flag leaf and the thirteenth leaf of 97-7. During reversible phase of photosynthetic declination,endopeptidase activity remained at a low level and increased slightly only in the later part of this phase. Then it rose up rapidly at irreversible decline phase and reached a very high level. For Wuyujing 3, the change in endopeptidase activity in the thirteenth leaf was parallel to that in flag leaf. However, for 97-7, the rapid increase of endopeptidase activity in the thirteenth leaf started later than that of flag leaf. The results implied that the rate of protein breakdown and conversion to transportable nitrogen in leaves of 97-7 was slower than that in leaves of Wuyujing 3 during photosynthetic declination and it led to relatively lower seed setting rate and fully filling grains rate in 97-7. This may be one of the important reasons why 97-7 could not bring the high yield potentiality into play and the findings may be taken into consideration while breeding for high potential varieties in future.

  10. Insulin aspart pharmacokinetics

    DEFF Research Database (Denmark)

    Rasmussen, Christian Hove; Roge, Rikke Meldgaard; Ma, Zhulin;

    2014-01-01

    Background: Insulin aspart (IAsp) is used by many diabetics as a meal-time insulin to control postprandial glucose levels. As is the case with many other insulin types, the pharmacokinetics (PK), and consequently the pharmacodynamics (PD), is associated with clinical variability, both between...... to investigate and quantify the properties of the subcutaneous depot. Data from Brange et al. (1990) are used to determine the effects of insulin chemistry in subcutis on the absorption rate. Intravenous (i.v.) bolus and infusion PK data for human insulin are used to understand and quantify the systemic...... distribution and elimination (Porksen et al., 1997; Sjostrand et al., 2002). PK and PD profiles for type 1 diabetics from Chen et al. (2005) are analyzed to demonstrate the effects of IAsp antibodies in terms of bound and unbound insulin. PK profiles from Thorisdottir et al. (2009) and Ma et al. (2012b...

  11. Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity.

    Science.gov (United States)

    Dando, P M; Fortunato, M; Smith, L; Knight, C G; McKendrick, J E; Barrett, A J

    1999-01-01

    Legumain was recently discovered as a lysosomal endopeptidase in mammals [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098], having been known previously only from plants and invertebrates. It has been shown to play a key role in processing of the C fragment of tetanus toxin for presentation by the MHC class-II system [Manoury, Hewitt, Morrice, Dando, Barrett and Watts (1998) Nature (London) 396, 695-699]. We examine here the specificity of the enzyme from pig kidney by use of protein, oligopeptide and synthetic arylamide substrates, all determinations being made at pH 5.8. In proteins, only about one in ten of the asparaginyl bonds were hydrolysed, and these were mostly predicted to be located at turns on the protein surface. Bonds that were not cleaved in tetanus toxin were cleaved when presented in oligopeptides, sometimes faster than an equivalent oligopeptide based on a bond that was cleaved in the protein. Legumain cleaved the bait region of rat alpha1-macroglobulin and was 'trapped' by the macroglobulin, as most other endopeptidases are, but did not interact with human alpha2-macroglobulin, which contains no asparagine residue in its bait region. Glycosylation of asparagine totally prevented hydrolysis by legumain. Specificity for arylamide substrates was evaluated with reference to benzyloxycarbonyl-Ala-Ala-Asn-aminomethylcoumarin, and the preference for the P3-position amino acid was Ala>Tyr(tertiary butyl)>Val>Pro>Phe=Tyr>Leu=Gly. There was no hydrolysis of substrate analogues containing mono- or di-N-methylasparagines, l-2-amino-3-ureidopropionic acid or citrulline in the P1 position. We conclude that mammalian legumain appears to be totally restricted to the hydrolysis of asparaginyl bonds in substrates of all kinds. There seem to be no strong preferences for particular amino acids in other subsites, and yet there are still unidentified factors that prevent hydrolysis of many asparaginyl bonds

  12. Opioids, Neutral Endopeptidase, its Inhibitors and Cancer: Is There a Relationship among them?

    OpenAIRE

    Mizerska-Dudka, Magdalena; Kandefer-Szerszeń, Martyna

    2014-01-01

    The role of endogenous animal opioids in the biology of cancer is widely recognized but poorly understood. This is, among others, because of the short half-life of these peptides, which are quickly inactivated by endopeptidases, e.g., neutral endopeptidase (NEP, CD10). It has been established that NEP is engaged in the modulation of the tumor microenvironment, among others that of colon cancer, by exerting influence on cell growth factors, the extracellular matrix and other biologically activ...

  13. Separation and Characterization of Two Endopeptidases from Cotyledons of Germinating Vigna mungo Seeds.

    Science.gov (United States)

    Mitsuhashi, W; Koshiba, T; Minamikawa, T

    1986-03-01

    Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2-mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicatd that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts.

  14. Separation and Characterization of Two Endopeptidases from Cotyledons of Germinating Vigna mungo Seeds 1

    Science.gov (United States)

    Mitsuhashi, Wataru; Koshiba, Tomokazu; Minamikawa, Takao

    1986-01-01

    Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2-mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicatd that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts. Images Fig. 2 Fig. 3 Fig. 6 Fig. 7 PMID:16664675

  15. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2003-05-01

    Full Text Available Abstract Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102, only the bidirectional (Synechocystis PCC 6803 or both NiFe-hydrogenases (Anabaena PCC 7120 prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41% to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known

  16. CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM WOLFIPORIA COCOS

    Directory of Open Access Journals (Sweden)

    Katharina Cierpka

    2014-01-01

    Full Text Available As requirements for Advanced Therapy Medicinal Product (ATMP production differ from other production processes (e.g., therapeutic protein production, cell detachment is often a crucial step for the process success. In most cases, cell detachment is done enzymatically. Although many peptidases are established in cell culture in R&D, e.g., Trypsin as gold standard, many of them seem to be unsuitable in ATMP production processes. Therefore, the present study investigated a novel endopeptidase used in food biotechnology for its applicability in ATMP processes where cell detachment is needed. The Prolyl-specific Peptidase (PsP is of non-mammalian origin and considered as safe for humans. PsP was purified from the supernatant of the fungus Wolfiporia cocos. The isolation and purification resulted in an enzyme solution with 0.19 U mg-1 prolyl-specific activity. By in silico analysis it was confirmed that attachment-promoting proteins can be cleaved by PsP in a similar amount than with Trypsin. Further the proteolytic activity was determined for PsP and Trypsin by using the same enzymatic assay. Detachment with both enzymes was compared for cells used in typical therapeutic production processes namely a mesenchymal stem cell line (hMSC-TERT as a model for a cell therapeutic, Vero and MA104 cells used for viral therapeutic or vaccine production. The cell detachment experiments were performed with comparable enzyme activities (1.6 U mL-1. hMSC-TERT detachment was faster with PsP than with Trypsin. For Vero cells the detachment with PsP was not only faster but also more efficient. For MA104 cells the detachment rate with PsP was similar to Trypsin. For all cell types, detachment with PsP showed less influence on cell growth and metabolism compared to standard Trypsin.Thus, three cell types used in ATMP, viral therapeutics or vaccine production can be detached efficiently and gently with PsP. Therefore, PsP shows

  17. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    DEFF Research Database (Denmark)

    Wong, Jaslyn E M M; Midtgaard, Søren Roi; Gysel, Kira;

    2015-01-01

    of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering...

  18. Pathological release of urinary endopeptidase 24.11 early after renal transplantation.

    Science.gov (United States)

    Nortier, J; Abramowicz, D; Kinnaert, P; Deschodt-Lanckman, M; Vanherweghem, J L

    1992-01-01

    Endopeptidase 24.11 (EC 3.4.24.11) enzymatic activity was spectrofluorimetrically measured in human urine, using a synthetic peptidic substrate. Urinary endopeptidase 24.11 output (Uendo) was determined in 24-hour urine samples of 10 kidney transplant recipients during the first 2 weeks after surgery. In 9 patients, a large increase in Uendo levels was noted during the 1st and/or the 2nd postoperative days (mean +/- SEM of peak Uendo 624 +/- 122 micrograms/24 h, p = 0.0003 as compared to 239 +/- 20 micrograms/24 h in a healthy control population). This occurred whether patients received OKT3 (n = 6) or cyclosporine A (n = 3) as primary immunosuppression. Uendo returned to normal between the 3rd and the 5th postoperative day. We conclude that renal transplantation is associated with an early and marked release of endopeptidase 24.11 in urine. This could be due to the potentially toxic effects of ischemia and/or immunosuppressive drugs on the proximal tubular epithelium. The clinical usefulness of urinary endopeptidase 24.11 as a marker of tubular injury remains to be assessed. PMID:1308855

  19. Concurrent neutral endopeptidase and ACE inhibition in experimental heart failure: renal and hormonal effects

    DEFF Research Database (Denmark)

    Helin, K

    1993-01-01

    Neutral endopeptidase (NEP) inhibitors have been shown to strengthen the effects of endogenous atrial natriuretic peptide (ANP). It has been well documented that angiotensin I-converting enzyme (ACE) inhibitors act beneficially in chronic congestive heart failure (CHF). In the present study, renal...

  20. Chromosomal Location by Use of Trisomics and New Alleles of an Endopeptidase in Zea Mays

    DEFF Research Database (Denmark)

    Nielsen, Gunnar Gissel; Scandalios, John G.

    1974-01-01

    An association was found earlier between the Ep1 gene locus coding for an endopeptidase and the endosperm color gene Y1 on chromosome 6 of Zea mays. By employing primary trisomics we have unequivocally placed the Ep1 gene on chromosome 6, closely linked to the Y1 locus. Additionally we describe new...

  1. Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds.

    Science.gov (United States)

    Mitsuhashi, W; Minamikawa, T

    1989-01-01

    A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 +/- 1 degrees C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.

  2. Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds 1

    Science.gov (United States)

    Mitsuhashi, Wataru; Minamikawa, Takao

    1989-01-01

    A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 ± 1°C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:16666526

  3. Endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced Senescence

    Institute of Scientific and Technical Information of China (English)

    Peng Zhang; Fei Wang; Lie-Feng Zhang; Qi Rui; Lang-Lai Xu

    2007-01-01

    The changes and characteristics of endopeptidase (EP) isoenzymes in cucumber (Cucumis sativus L.) leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis (G-PAGE) containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes (CEP) with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs (CEP5, 6, 7 and CEP8) whose activities increased with dark-induced time were only detected in senescent leaves.Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 ℃ to 45 ℃, and the optimum temperature was found to be 40 ℃. However, the activities of CEPs were not detected below 25 ℃ or over 60 ℃. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2,3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescencerelated endopeptidases (CEP1, 5, 6, 7, 8 and 9) were found in cucumber leaves.

  4. The Botrytis cinerea aspartic proteinase family

    NARCIS (Netherlands)

    Have, ten A.; Espino, J.J.; Dekkers, E.; Sluyter, Van S.; Brito, N.; Kay, J.; González, C.; Kan, van J.A.L.

    2010-01-01

    The ascomycete plant pathogen Botrytis cinerea secretes aspartic proteinase (AP) activity. Functional analysis was carried out on five aspartic proteinase genes (Bcap1-5) reported previously. Single and double mutants lacking these five genes showed neither a reduced secreted proteolytic activity, n

  5. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

    Directory of Open Access Journals (Sweden)

    Mareš Michael

    2008-03-01

    Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

  6. Screening of aspartate dehydrogenase of bacteria

    OpenAIRE

    Fukuda, Shoko; Okamura, Tokumitsu; Yasumasa, Izumi; Takeno, Tomomi; Ohsugi, Masahiro

    2001-01-01

    Fifty-two strains of bacteria cultured under aerobic conditions and 12 strains of bacteria cultured under anaerobic conditions demonstrated high activity staining of aspartate dehydrogenase with NAD^+. Four strains of bacteria cultured under aerobic conditions and 7 strains of bacteria cultured under anaerobic conditions demonstrated high activity staining of aspartate dehydrogenase with NADP^+. Seven strains of bacteria cultured under aerobic conditions and 4 strains of bacteria cultured und...

  7. Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity

    OpenAIRE

    Shan, Lu; Mathews, Irimpan I.; Khosla, Chaitan

    2005-01-01

    Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-Å resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus ...

  8. Glycyl endopeptidase from papaya latex: partial purification and use for production of fish gelatin hydrolysate.

    Science.gov (United States)

    Karnjanapratum, Supatra; Benjakul, Soottawat

    2014-12-15

    An aqueous two-phase system (ATPS) in combination with ammonium sulphate ((NH4)2SO4) precipitation was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH4)2SO4 and MgSO4) at different concentrations were used. Glycyl endopeptidase with high purification fold (PF) and yield was found in the salt-rich bottom phase of ATPS with 10%PEG 6000-10% (NH4)2SO4. When ATPS fraction from Red Lady cultivar was further precipitated with 40-60% saturation of (NH4)2SO4, PF of 2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable odour could be prepared with the aid of PPGE. PMID:25038693

  9. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Jaslyn E. M. M. [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Midtgaard, Søren Roi [University of Copenhagen, Universitetsparken 5, 2100 Copenhagen (Denmark); Gysel, Kira [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J. [University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Stougaard, Jens; Thirup, Søren; Blaise, Mickaël, E-mail: mickael.blaise@cpbs.cnrs.fr [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark)

    2015-03-01

    The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  10. Dataset of cocoa aspartic protease cleavage sites.

    Science.gov (United States)

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  11. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

    Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.

  12. Computer-aided lead optimization: improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A.

    Directory of Open Access Journals (Sweden)

    Jing Tang

    Full Text Available Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i (app of 7+/-2.4 microM using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i (app of 3.8+/-0.8 microM with a relatively small increase in molecular weight (16 Da. The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.

  13. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    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.

  14. Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae).

    Science.gov (United States)

    Pardo, M F; López, L M; Canals, F; Avilés, F X; Natalucci, C L; Caffini, N O

    2000-09-01

    A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme. The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

  15. Peptidase Activities of Tripeptidyl Peptidase Ⅰ(TPP Ⅰ): Exopeptidase and Endopeptidase

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The defect of TPP Ⅰ causes a disease, late infantile neuronal ceroid lipofuscinosis(LINCL, CLN2). To investigate the bio-activity of tripeptidyl peptidase Ⅰ(TPP Ⅰ) from rat kidneys, the effects of digestion of angiotensin Ⅱ(Ang Ⅱ) and a synthetic endo-type substrate(Gly1-Lys-Pro-Iie-Pro5-Phe-Phe-Arg-Leu-Lys10) via TPP Ⅰ were analyzed by HPLC and TOF-MS. The data suggest that the degradation rate of Ang Ⅱ can reach 18.2% by the rat TPP Ⅰ and DRV(Asp-Arg-Val) can be released from N-termini of Ang Ⅱ within 16 h. In addition, the synthetic endo-type substrate is cleaved at the same position between Phe6 and Phe7. Accordingly, TPP Ⅰ shows two kinds of peptidase activities. One is a tripeptidyl peptidase activity and the other is a pepstatin insensitive carboxyl endopeptidase activity. Tripeptidyl peptidase activity and pepstatin insensitive carboxyl endopeptidase activity seem to be dual phases of one enzyme, TPP Ⅰ.

  16. CLONING AND SEQUENCING OF THE GENE FOR A LACTOCOCCAL ENDOPEPTIDASE, AN ENZYME WITH SEQUENCE SIMILARITY TO MAMMALIAN ENKEPHALINASE

    NARCIS (Netherlands)

    Mierau, Igor; Tan, Paris S.T.; Haandrikman, Alfred J.; Kok, Jan; Leenhouts, Kees J.; Konings, Wil N.; Venema, Gerard

    1993-01-01

    The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-247 in lambdaEMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport syste

  17. D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons

    DEFF Research Database (Denmark)

    Gong, Xiang-Qun; Frandsen, Anne; Lu, Wei-Yang;

    2005-01-01

    to the right. Schild plot analysis indicated that D-aspartate acts competitively to block AMPARs. The K(b) for D-aspartate was estimated to be 0.93 mM. 4 D-aspartate also blocked L-glutamate-induced current in Xenopus laevis oocytes that expressed recombinant homomeric AMPARs. 5 NMDA possessed similar...

  18. Solution Structure of IseA, an Inhibitor Protein of DL-Endopeptidases from Bacillus subtilis, Reveals a Novel Fold with a Characteristic Inhibitory Loop

    OpenAIRE

    Arai, Ryoichi; Fukui, Sadaharu; Kobayashi, Naoya; Sekiguchi, Junichi

    2012-01-01

    In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determ...

  19. Structural basis for type VI secreted peptidoglycan dl-endopeptidase function, specificity and neutralization in Serratia marcescens

    Energy Technology Data Exchange (ETDEWEB)

    Srikannathasan, Velupillai; English, Grant [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Bui, Nhat Khai [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Trunk, Katharina; O’Rourke, Patrick E. F.; Rao, Vincenzo A. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Vollmer, Waldemar [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Coulthurst, Sarah J., E-mail: s.j.coulthurst@dundee.ac.uk; Hunter, William N., E-mail: s.j.coulthurst@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2013-12-01

    Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2

  20. Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

    NARCIS (Netherlands)

    Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.

    2008-01-01

    The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not a

  1. Molecular cloning and characterization of Vigna mungo processing enzyme 1 (VmPE-1), an asparaginyl endopeptidase possibly involved in post-translational processing of a vacuolar cysteine endopeptidase (SH-EP).

    Science.gov (United States)

    Okamoto, T; Minamikawa, T

    1999-01-01

    Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar cysteine endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.

  2. Purification and characterization of four new cysteine endopeptidases from fruits of Bromelia pinguin L. grown in Cuba.

    Science.gov (United States)

    Payrol, Juan Abreu; Obregón, Walter D; Trejo, Sebastián A; Caffini, Néstor O

    2008-02-01

    Bromelia pinguin L. is a plant broadly distributed in Central America and Caribbean islands. The fruits have been used in traditional medicine as anthelmintic, probably owed to the presence of a mixture of cysteine endopeptidases, initially termed pinguinain. This work deals with the purification and characterization of the four main components of that mixture, two of them showing acid pI and the other two alkaline pI. Molecular masses (SDS-PAGE and MALDI-TOF), N-terminal sequence and the reactivity and kinetic parameters versus synthetic substrates (p-nitrophenyl-N-alpha-CBZ-amino acid esters, PFLNA, Z-Arg-Arg-p-NA, and Z-Phe-Arg-p-NA) of the studied peptidases are given, as well as the N-terminal sequences of the enzymes and the homology degree with other plant endopeptidases.

  3. NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

    International Nuclear Information System (INIS)

    The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, the authors have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models or prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substrates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. They propose that this structure (designated the CA knuckle) provides a recognition element for type II proinsulin endopeptidase

  4. NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, M.A. (Harvard Medical School, Boston, MA (USA) Massachusetts General Hospital, Boston (USA)); Frank, B.H.; Heiney, R.; Pekar, A. (Eli Lilly and Co., Indianapolis, IN (USA)); Khait, I.; Neuringer, L.J. (Massachusetts Institute of Technology, Cambridge (USA)); Shoelson, S.E. (Harvard Medical School, Boston (USA))

    1990-09-11

    The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg{sup 31}-Arg{sup 32}) and a type II activity that cleaves the C-peptide/A-chain junction (Lys{sup 64}-Arg{sup 65}). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, the authors have undertaken comparative {sup 1}H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models or prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the {sup 1}H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substrates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic {sup 1}H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. They propose that this structure (designated the CA knuckle) provides a recognition element for type II proinsulin endopeptidase.

  5. Medial temporal N-acetyl-aspartate in pediatric major depression.

    Science.gov (United States)

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  6. Medial temporal N-acetyl aspartate in pediatric major depression

    Science.gov (United States)

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  7. Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

    Science.gov (United States)

    Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit

    2016-03-01

    Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

  8. Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation.

    Directory of Open Access Journals (Sweden)

    Sophia Maschalidi

    Full Text Available Intracellular Toll-like receptors (TLRs expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV, a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8(+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.

  9. Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation.

    Science.gov (United States)

    Maschalidi, Sophia; Hässler, Signe; Blanc, Fany; Sepulveda, Fernando E; Tohme, Mira; Chignard, Michel; van Endert, Peter; Si-Tahar, Mustapha; Descamps, Delphyne; Manoury, Bénédicte

    2012-01-01

    Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8(+) T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases. PMID:22916010

  10. Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate.

    Science.gov (United States)

    Hsieh, Cheng-Hong; Wang, Tzu-Yuan; Hung, Chuan-Chuan; Hsieh, You-Liang; Hsu, Kuo-Chiang

    2016-01-01

    Prolyl endopeptidase (PEP) has been associated with neurodegenerative disorders, and the PEP inhibitors can restore the memory loss caused by amnesic compounds. In this study, we investigated the PEP inhibitory activity of the enzymatic hydrolysates from various food protein sources, and isolated and identified the PEP inhibitory peptides. The hydrolysate obtained from sodium caseinate using bromelain (SC/BML) displayed the highest inhibitory activity of 86.8% at 5 mg mL(-1) in the present study, and its IC50 value against PEP was 0.77 mg mL(-1). The F-5 fraction by RP-HPLC (reversed-phase high performance liquid chromatography) from SC/BML showed the highest PEP inhibition rate of 88.4%, and 9 peptide sequences were identified. The synthetic peptides (1245.63-1787.94 Da) showed dose-dependent inhibition effects on PEP as competitive inhibitors with IC50 values between 29.8 and 650.5 μM. The results suggest that the peptides derived from sodium caseinate have the potential to be PEP inhibitors. PMID:26574880

  11. Aspartate inhibits Staphylococcus aureus biofilm formation.

    Science.gov (United States)

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp.

  12. Aspartate inhibits Staphylococcus aureus biofilm formation.

    Science.gov (United States)

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. PMID:25687923

  13. In silico methods to identify meat-derived prolyl endopeptidase inhibitors.

    Science.gov (United States)

    Lafarga, Tomas; O'Connor, Paula; Hayes, Maria

    2015-05-15

    According to the World Health Organization (WHO), approximately 450 million people suffer from mental or neurological disorders and five of the ten leading causes of disability and premature death worldwide are psychiatric conditions. Social, biological and neurological sciences provided extensive understanding into the role of risk and protective factors in the development of mental disorders and poor mental health. Altered activity of a number of enzymes, such as prolyl endopeptidase (PEP, EC 3.4.21.26), has been linked to the prevention and treatment of a number of mental disorders, including anxiety, depression and Alzheimer's disease. The inhibition of PEP has potential for use in the prevention and in the treatment of mental disorders. The objective of this work was to identify PEP-inhibitory peptides from meat proteins using in silico methods. In this paper, five proteins commonly found in meat by-products were evaluated as a substrate for use in the generation of PEP inhibitory peptides. These include serum albumin, collagen and myosin. These proteins were cleaved in silico using BIOPEP and ExPASy PeptideCutter and the generated peptides were compared to known PEP-inhibiting peptides in the database of BIOPEP. A number of novel PEP inhibitory peptide sequences were identified in this study, including PPL, APPH, IPP and PPG with corresponding IC50 values of 2.86, 3.95, 4.02 and 2.70 mM, respectively. This work demonstrates the usefulness of in silico analysis for predicting the release of PEP-inhibiting peptides from meat proteins.

  14. Schistosome asparaginyl endopeptidase (legumain) is not essential for cathepsin B1 activation in vivo.

    Science.gov (United States)

    Krautz-Peterson, Greice; Skelly, Patrick J

    2008-05-01

    Schistosomes are parasitic platyhelminths that constitute an important public health problem. Adult parasites live in the vasculature of their vertebrate hosts where they consume blood. Ingested blood proteins are degraded by a proteolytic cascade. One of the best characterized schistosome proteases is cathepsin B1 (SmCB1 or Sm31). This protein is synthesized as a large 38 kDa precursor form which is proteolytically cleaved to yield a mature, active 31 kDa enzyme. A second schistosome protease--the asparaginyl endopeptidase SmAE (also known as Sm32, or schistosome legumain), has been proposed to proteolytically convert the 38 kDa precursor SmCB1 into its mature form. Recombinant activated SmAE has been shown to trans-process SmCB1 into the mature, catalytic form in vitro. In the present study, our aim was to test the hypothesis that in vivo SmAE likewise processes SmCB1 into its active form. To do this, expression of the SmAE gene was suppressed in adult Schistosoma mansoni using RNA interference (RNAi). The results of these experiments show that, even in the absence of detectable SmAE protein, SmCB1 is fully processed and active and support the assertion that SmAE is not essential to activate SmCB1 in vivo. The data indicate that our original hypothesis is incorrect and that SmAE is not pivotal in the in vivo conversion of cathepsin B1 into its mature, active form.

  15. Renal Actions of Neutral Endopeptidase Inhibition in Rats with Chronic Heart Failure

    Directory of Open Access Journals (Sweden)

    Amr M. Abbas

    2010-01-01

    Full Text Available Problem statement: We aim to evaluate the effects of acute and chronic inhibition of Neutral EndoPeptidase (NEP, by ONO-9902, on plasma and renal NEP gene expression, hemodynamic and renal parameters in rats with Chronic Heart Failure (CHF following left Coronary Artery Ligation (CAL. Approach: Forty eight male Sprague-Dawley rats (220-240 g were divided into sham and CAL groups. Myocardial infarction was induced by left CAL. All rats were further subdivided into untreated and orally treated with ONO-9902 (300 mg kg-1 day-1 from the 1st to 6th weeks after the operation. At the 1st and 6th weeks after the operation, gene expression of plasma and renal NEP, plasma ANP, cGMP and aldosterone concentrations, urine volume, Na and ANP excretion, creatinine clearance and renal cGMP generation were measured. Results: CAL leads to sodium and water retention, increased renal NEP gene expression, plasma ANP and aldosterone and decreased renal cGMP generation and plasma NEP gene expression. Acute treatment of CAL rats by ONO-9902, at the 1st week after the operation, inhibited plasma and renal NEP gene expression with increased plasma ANP, which caused diuresis, natriuresis and increased renal cGMP generation. Moreover, chronic treatment of those rats by ONO-9902 decreased plasma and renal NEP gene expression, plasma aldosterone, increased plasma ANP but non significantly, and caused diuresis, natriuresis with increased renal cGMP generation. GFR was not significantly changed before or after treatment. Conclusion: Chronic treatment with NEP inhibitor decreases Na and water retention in rats with CHF by enhancing ANP action and suppressing aldosterone secretion. So, ONO-9902 may offer a new therapeutic approach in patients with CHF.

  16. Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea.

    Science.gov (United States)

    Griggs, David W; Prinsen, Michael J; Oliva, Jonathan; Campbell, Mary A; Arnett, Stacy D; Tajfirouz, Deena; Ruminski, Peter G; Yu, Ying; Bond, Brian R; Ji, Yuhua; Neckermann, Georg; Choy, Robert K M; de Hostos, Eugenio; Meyers, Marvin J

    2016-05-01

    Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy. PMID:26907621

  17. Structural and mechanistic analysis of two prolyl endopeptidases: role of interdomain dynamics in catalysis and specificity.

    Science.gov (United States)

    Shan, Lu; Mathews, Irimpan I; Khosla, Chaitan

    2005-03-01

    Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-A resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus xanthus was crystallized (1.5-A resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features. PMID:15738423

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

    Science.gov (United States)

    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.

  19. Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

    Science.gov (United States)

    Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai; Trunk, Katharina; O'Rourke, Patrick E F; Rao, Vincenzo A; Vollmer, Waldemar; Coulthurst, Sarah J; Hunter, William N

    2013-12-01

    Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

  20. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

    Science.gov (United States)

    Kang, Chao; Yu, Xiao-Wei; Xu, Yan

    2015-02-01

    A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system. PMID:25547787

  1. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

    Science.gov (United States)

    Kang, Chao; Yu, Xiao-Wei; Xu, Yan

    2015-02-01

    A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.

  2. Aspartate aminotransferase – key enzyme in the human systemic metabolism

    Directory of Open Access Journals (Sweden)

    Dagmara Otto-Ślusarczyk

    2016-03-01

    Full Text Available Aspartate aminotransferase is an organ - nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms - cytoplasmic (AST1 and mitochondrial (AST2, that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys – 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs.

  3. Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Leung, F.Y.; Niblock, A.E.; Henderson, A.R.

    1984-08-01

    A description is given of the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. The antisera were partly purified by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

  4. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    Science.gov (United States)

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  5. Identification in Lupin Seed of a Serine-Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

    Institute of Scientific and Technical Information of China (English)

    Chiara Magni; Fabio Sessa; Gabriella Tedeschi; Armando Negri; Alessio Scarafoni; Alessandro Consonni; Marcello Duranti

    2012-01-01

    The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation.No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins.In this work,a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds.The endopeptidase was tested with a number of endogenous and exogenous protein substrates,which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences.The observed hydrolysis patterns were limited and highly specific.Partial proteolysis led to stable polypeptide fragments that were characterized by 1-and 2-D electrophoresis.Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses.These approaches,supported by bioinformatic analysis of the available sequences,allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates.The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF),leupeptin,and serine proteinase protein inhibitors,while it was not affected by pepstatin,transEpoxysuccinyI-L-leucylamido(4-guanidino)butane (E64),and ethylenediaminetetraacetic acid (EDTA),thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase.The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving-R-R-bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

  6. Aspartate Aminotransferase - Bridging Carbohydrate and Energy Metabolism in Plasmodium Falciparum

    NARCIS (Netherlands)

    Wrenger, Carsten; Mueller, Ingrid B.; Silber, Ariel M.; Jordanova, Rositsa; Lamzin, Victor S.; Groves, Matthew R.

    2012-01-01

    In this mini-review we briefly examine and summarize evidence on the role of the plasmodial aspartate aminotransferase (AspAT) of the malarial parasite. Recent data have provided information on the products of the purine salvage pathway as well as the glycolytic and oxidative phosphorylation pathway

  7. Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

    Science.gov (United States)

    Kobayashi, S; Millhorn, D E

    2000-01-01

    The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress. PMID:11113364

  8. Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis.

    OpenAIRE

    Shipp, M A; Vijayaraghavan, J.; Schmidt, E V; Masteller, E L; D'Adamio, L; Hersh, L.B.; Reinherz, E L

    1989-01-01

    The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopep...

  9. Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

    Science.gov (United States)

    Eugster, Philippe J; Salamin, Karine; Grouzmann, Eric; Monod, Michel

    2015-12-01

    Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus. PMID:26464108

  10. 2-(hydroxymethyl)aspartic acid: synthesis, crystal structure, and reaction with a transaminase

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, J.J.; Metzler, D.E.; Powell, D.; Jacobson, R.A.

    1980-11-05

    The synthesis and x-ray crystal structure of 2-(hydroxymethyl) aspartic acid and the preliminary evaluation of its interaction with cytosolic aspartate aminotransferase of pig heart are described. A dissociation constant 1.4 mM for the L-2-(hydroxymethyl) aspartate complex with the enzyme was obtained. 2 figures. (DP)

  11. Three-dimensional hybrid networks based on aspartic acid

    Indian Academy of Sciences (India)

    Anupama Ghosh; R A Sanguramath

    2008-01-01

    Three-dimensional achiral coordination polymers of the general formula M2(D, L-NHCH (COO)CH2COO)2.C4H4N2 where M = Ni and Co and pyrazine acts as the linker molecule have been prepared under hydrothermal conditions starting with [M(L-NHCH(COO)CH2COO).3H2O] possessing a helical chain structure. A three-dimensional hybrid compound of the formula Pb2.5[N{CH(COO)CH2COO}22H2O] has also been prepared hydrothermally starting with aspartic acid and Pb(NO3)2. In this lead compound, where a secondary amine formed by the dimerisation of aspartic acid acts as the ligand, there is two-dimensional inorganic connectivity and one-dimensional organic connectivity.

  12. A superactive insulin: [B10-aspartic acid]insulin(human).

    OpenAIRE

    Schwartz, G P; Burke, G. T.; Katsoyannis, P G

    1987-01-01

    The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-10 of the B chain of insulin. We have synthesized a human insulin analogue, [AspB10]insulin, corresponding to the mutant proinsulin and evaluated its biological activity. [AspB10]Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 +/- 14...

  13. In vivo multiplex quantitative analysis of 3 forms of alpha melanocyte stimulating hormone in pituitary of prolyl endopeptidase deficient mice

    Directory of Open Access Journals (Sweden)

    Perroud Bertrand

    2009-06-01

    Full Text Available Abstract Background In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP, a serine protease, on alpha melanocyte stimulating hormone (α-MSH, we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of α-MSH. Methods Using Multiple Reaction Monitoring (MRM, we analyzed peptide transitions to quantify three different forms of α-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile. Results We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (α-MSH1–13, by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (α-MSH1–12 product – the 12 residues α-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of α-MSH: the deacetylated α-MSH1–13, the acetylated α-MSH1–13 and the truncated form α-MSH1–12. For this experiment, we used a mouse model (PREP-GT in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated α-MSH1–13 and α-MSH1–12 is significantly increased (P-value = 0.015, N = 6 in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of α-MSH1–13 versus the deacetylated α-MSH1–13. These results combined with the demonstration that PREP digests α-MSH1–13 in vitro, strongly suggest that α-MSH1–13 is an in vivo substrate of PREP. Conclusion The multiplex targeted quantitative peptidomics technique we

  14. Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase.

    Science.gov (United States)

    Sajid, Mohammed; McKerrow, James H; Hansell, Elizabeth; Mathieu, Mary A; Lucas, Kimberley D; Hsieh, Ivy; Greenbaum, Doron; Bogyo, Matthew; Salter, Jason P; Lim, Kee C; Franklin, Christopher; Kim, Jea-Hyoun; Caffrey, Conor R

    2003-09-01

    Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.

  15. Vesicular uptake and exocytosis of l-aspartate is independent of sialin

    Science.gov (United States)

    Morland, Cecilie; Nordengen, Kaja; Larsson, Max; Prolo, Laura M.; Farzampour, Zoya; Reimer, Richard J.; Gundersen, Vidar

    2013-01-01

    The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of l-aspartate is independent of sialin. PMID:23221336

  16. Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

    Directory of Open Access Journals (Sweden)

    Braz Vânia S.

    2002-01-01

    Full Text Available The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1 encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3, which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30% of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function.

  17. Temporal changes in neutral endopeptidase/CD10 immunoexpression in the cyclic and early pregnant canine endometrium.

    Science.gov (United States)

    Payan-Carreira, R; Santos, C; Miranda, S; Pereira, R M L N; Santos, D; Pires, M A

    2014-10-01

    CD10 is a multifunctional transmembrane neutral endopeptidase (NEP) that is considered to be a reliable marker of ectopic human endometrial stroma. Available information on NEP/CD10 protein expression in animal endometria is scarce. This study focused on the immunolocalization of NEP/CD10 in the canine uterus and on its temporal changes during the estrous cycle and early pregnancy (Days 11 to 23 post-LH surge) in healthy females. NEP/CD10 expression was found in the canine endometrial stroma in all stages of the estrous cycle, showing cyclic differences both in intensity and in distribution pattern. A small population of negative stromal cells in subsurface position was also observed. This population shared some morphological characteristics with the human predecidual cells, which became positive in progesterone-associated stages of the cycle. In addition, positive immunolabeling was also observed in canine myometrial stroma. In early pregnancy, the basal glandular epithelia and the syncytium cords remained negative to this marker contrasting with the trophoblast and the lacunar epithelium. A weak to moderate intensity of immunolabeling was observed in the decidual cells, whereas stromal immunolabeling was more intense at the delimitation of the syncytium cords. In conclusion, CD10 is consistently expressed in the canine endometrial stroma and myometrium but not in the endometrial epithelia. The characteristic pattern seen in early pregnancy also suggests a role for this molecule in the process of embryo invasion at implantation. PMID:25082021

  18. Comparison of a soluble co-formulation of insulin degludec/insulin aspart vs biphasic insulin aspart 30 in type 2 diabetes

    DEFF Research Database (Denmark)

    Niskanen, Leo; Leiter, Lawrence A; Franek, Edward;

    2012-01-01

    Insulin degludec/insulin aspart (IDegAsp) is a soluble co-formulation of insulin degludec (70%) and insulin aspart (IAsp: 30%). Here, we compare the efficacy and safety of IDegAsp, an alternative IDegAsp formulation (AF: containing 45% IAsp), and biphasic IAsp 30 (BIAsp 30)....

  19. Aspartic proteinases in the digestive tract of marine decapod crustaceans.

    Science.gov (United States)

    Navarrete del Toro, María de Los Angeles; García-Carreño, Fernando; López, Manuel Díaz; Celis-Guerrero, Laura; Saborowski, Reinhard

    2006-08-01

    Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others. PMID:16788916

  20. Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

    Science.gov (United States)

    Čulić, Ognjen; Cordero, Mario D; Žanić-Grubišić, Tihana; Somborac-Bačura, Anita; Pučar, Lara Batičić; Detel, Dijana; Varljen, Jadranka; Barišić, Karmela

    2016-10-01

    Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

  1. Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae.

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    Gaurab Sircar

    Full Text Available Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation 'Rhi o 1'.The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross

  2. Dual neural endopeptidase/endothelin-converting [corrected] enzyme inhibition improves endothelial function in mesenteric resistance arteries of young spontaneously hypertensive rats

    DEFF Research Database (Denmark)

    Lemkens, Pieter; Nelissen, Jelly; Meens, Merlijn J P M T;

    2012-01-01

    BACKGROUND: Endothelin-1 (ET1) is a potent vasoconstrictor peptide with pro-mitogenic and pro-inflammatory properties and is therefore of interest in the development of endothelial dysfunction, endothelium-dependent flow-related remodeling, and hypertension-related remodeling. ET1 can be formed...... through cleavage of big ET1 by endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP). METHOD: We investigated whether the dual NEP/ECE inhibitor SOL1 improves resistance artery function and structure in 12 weeks old spontaneously hypertensive rats (SHRs) and whether arterial structural...

  3. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    Science.gov (United States)

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that

  4. Preparation and enantiosorption of L-aspartic acid pillared hydrotalcites

    Institute of Scientific and Technical Information of China (English)

    PENG Xia-hui; HUANG Ke-long

    2007-01-01

    L-aspartic acid (Asp) pillared hydrotalcites were prepared by direct reaction of the L-Asp anion with layered double hydroxides (LDHs). The obtained samples were characterized by X-ray diffractometry (XRD), Fourier transform infrared (FTIR),and thermogravimetric and differential thermal analysis (TG/DTA). The results show that the initial interlayer carbonate ions can be completely replaced by the L-Asp anion under the controlled conditions. The pillared hydrotalcites have a crystallized supramolecular structure and thermal stability. The L-Asp pillared LDHs were used in the enantiosorption of enantiopure phenylalanine (Phe), the results suggest that L-Asp pillared LDHs exhibit an excellent enantiosorption capability for D-Phe, and the adsorption isotherm fits Freundlich equation.

  5. N-methyl-D-aspartic acid receptor agonists

    DEFF Research Database (Denmark)

    Madsen, U; Frydenvang, Karla Andrea; Ebert, B;

    1996-01-01

    (R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy......) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly...

  6. Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

    Science.gov (United States)

    Wales, M E; Madison, L L; Glaser, S S; Wild, J R

    1999-12-17

    The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity

  7. Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1 is involved in pathogen defense

    Directory of Open Access Journals (Sweden)

    Timo eHöwing

    2014-02-01

    Full Text Available Programmed cell death (PCD is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP with a C-terminal KDEL endoplasmic reticulum (ER retention signal (KDEL CysEP. KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3 are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodelling.We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.

  8. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    Science.gov (United States)

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  9. Properties of Copolymers of Aspartic Acid and Aliphatic Dicarboxylic Acids Prepared by Reactive Extrusion

    Science.gov (United States)

    Aspartic acid may be prepared chemically or by the fermentation of carbohydrates. Currently, low molecular weight polyaspartic acids are prepared commercially by heating aspartic acid at high temperatures (greater than 220 degrees C) for several hours in the solid state. In an effort to develop a ...

  10. Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds.

    Science.gov (United States)

    Hansen, Alexandar L; Behrman, Edward J

    2016-08-01

    We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid. PMID:27258673

  11. Reversible helix sense inversion in surface-grafted poly(beta-phenethyl-L-aspartate) films

    NARCIS (Netherlands)

    Luijten, Jeroen; Vorenkamp, Eltjo J.; Schouten, Arend J.

    2007-01-01

    The reversible manipulation of the helix screw sense in surface-grafted poly(beta-phenethyl-L-aspartate) (PPELA) films by means of external stimuli was investigated. Ringopening polymerization of beta-phenethyl-L-aspartate N-carboxyanhydride initiated from primary amino-functionalized silicon and qu

  12. Reversible Helix Sense Inversion in Surface-Grafted Poly(β-phenethyl-L-aspartate) Films

    NARCIS (Netherlands)

    Luijten, Jeroen; Vorenkamp, Eltjo J.; Schouten, Arend J.

    2007-01-01

    The reversible manipulation of the helix screw sense in surface-grafted poly(β-phenethyl-L-aspartate) (PPELA) films by means of external stimuli was investigated. Ringopening polymerization of β-phenethyl-L-aspartate N-carboxyanhydride initiated from primary amino-functionalized silicon and quartz s

  13. Superactive insulin: [B10-aspartic acid]insulin(human)

    International Nuclear Information System (INIS)

    The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-10 of the B chain of insulin. The authors have synthesized a human insulin analogue, [Asp/sup B10/] insulin, corresponding to the mutant proinsulin and evaluated its biological activity. [Asp/sup B10/] Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 +- 146% relative to the natural hormone. In lipogenesis assays, the synthetic analogue exhibited a potency that was 435 +- 144% relative to insulin, which is statistically not different from its binding affinity. Reversed-phase HPLC indicated that the synthetic analogue is more apolar than natural insulin. They suggest that the observed properties reflect changes in the conformation of the analogue relative to natural insulin, which results in a stronger interaction with the insulin receptor. Thus, a single substitution of an amino acid residue of human insulin has resulted in a superactive hormone

  14. Superactive insulin: (B10-aspartic acid)insulin(human)

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, G.P.; Burke, G.T.; Katsoyannis, P.G.

    1987-09-01

    The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-10 of the B chain of insulin. The authors have synthesized a human insulin analogue, (Asp/sup B10/) insulin, corresponding to the mutant proinsulin and evaluated its biological activity. (Asp/sup B10/) Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 +- 146% relative to the natural hormone. In lipogenesis assays, the synthetic analogue exhibited a potency that was 435 +- 144% relative to insulin, which is statistically not different from its binding affinity. Reversed-phase HPLC indicated that the synthetic analogue is more apolar than natural insulin. They suggest that the observed properties reflect changes in the conformation of the analogue relative to natural insulin, which results in a stronger interaction with the insulin receptor. Thus, a single substitution of an amino acid residue of human insulin has resulted in a superactive hormone.

  15. Solution structure of IseA, an inhibitor protein of DL-endopeptidases from Bacillus subtilis, reveals a novel fold with a characteristic inhibitory loop.

    Science.gov (United States)

    Arai, Ryoichi; Fukui, Sadaharu; Kobayashi, Naoya; Sekiguchi, Junichi

    2012-12-28

    In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3(10)-helix, and eight β-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between β4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes. PMID:23091053

  16. Solution Structure of IseA, an Inhibitor Protein of dl-Endopeptidases from Bacillus subtilis, Reveals a Novel Fold with a Characteristic Inhibitory Loop*

    Science.gov (United States)

    Arai, Ryoichi; Fukui, Sadaharu; Kobayashi, Naoya; Sekiguchi, Junichi

    2012-01-01

    In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, dl-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the dl-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation dl-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 310-helix, and eight β-strands, which is a novel fold like a “hacksaw.” Noteworthy is a dynamic loop between β4 and the 310-helix, which resembles a “blade.” The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4–310 loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4–310 loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes. PMID:23091053

  17. The effect of postirradiation application of aspartic acid salts on hemopoietic recovery in sublethally X-irradiated mice

    International Nuclear Information System (INIS)

    The effect of aspartic acid salts, especially of K and Mg aspartates, on certain hematological changes in the peripheral blood and hemopoietic organs of sublethally X-irratiated male mice of the strain C57Bl/10 was investigated. Salts of aspartic acid were administered in tap water after irradiation. A favorable effect of aspartic acid salts on erythropoietic recovery and on regeneration of thymus weight was found during the first two weeks after irradiation. (orig.)

  18. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Kolkata cohort of the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    Anirban Majumder

    2013-01-01

    Full Text Available Background: The A 1 chieve, a multicentric (28 countries, 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726 in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Kolkata, India. Results: A total of 576 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 417, insulin detemir (n = 70, insulin aspart (n = 55, basal insulin plus insulin aspart (n = 19 and other insulin combinations (n = 15. At baseline, glycaemic control was poor for both insulin naïve (mean HbA 1 c: 8.3% and insulin user (mean HbA 1 c: 8.6% groups. After 24 weeks of treatment, both the groups showed improvement in HbA 1 c (insulin naïve: −1.3%, insulin users: −1.4%. SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  19. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Hyderabad cohort of the A1chieve study

    Science.gov (United States)

    Santosh, R.; Mehrotra, Ravi; Sastry, N. G.

    2013-01-01

    Background: The A1chieve, a multicentric (28 countries), 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726) in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Hyderabad, India. Results: A total of 1249 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 893), insulin detemir (n = 158), insulin aspart (n = 124), basal insulin plus insulin aspart (n = 19) and other insulin combinations (n = 54). At baseline glycaemic control was poor for both insulin naïve (mean HbA1c: 9.0%) and insulin user (mean HbA1c: 9.5%) groups. After 24 weeks of treatment, both the groups showed improvement in HbA1c (insulin naïve: −0.9%, insulin users: −1.1%). SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. PMID:24404501

  20. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp YM55-1

    NARCIS (Netherlands)

    Veetil, Vinod Puthan; Raj, Hans; Quax, Wim J.; Janssen, Dick B.; Poelarends, Gerrit J.

    2009-01-01

    Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of l-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate.

  1. Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

    Science.gov (United States)

    Panda, Rakhi; Fiedler, Katherine L; Cho, Chung Y; Cheng, Raymond; Stutts, Whitney L; Jackson, Lauren S; Garber, Eric A E

    2015-12-01

    The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

  2. Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

    Science.gov (United States)

    Panda, Rakhi; Fiedler, Katherine L; Cho, Chung Y; Cheng, Raymond; Stutts, Whitney L; Jackson, Lauren S; Garber, Eric A E

    2015-12-01

    The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten. PMID:26548701

  3. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    OpenAIRE

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of ...

  4. Chiral Asymmetric Structures in Aspartic Acid and Valine Crystals Assessed by Atomic Force Microscopy.

    Science.gov (United States)

    Teschke, Omar; Soares, David Mendez

    2016-03-29

    Structures of crystallized deposits formed by the molecular self-assembly of aspartic acid and valine on silicon substrates were imaged by atomic force microscopy. Images of d- and l-aspartic acid crystal surfaces showing extended molecularly flat sheets or regions separated by single molecule thick steps are presented. Distinct orientation surfaces were imaged, which, combined with the single molecule step size, defines the geometry of the crystal. However, single molecule step growth also reveals the crystal chirality, i.e., growth orientations. The imaged ordered lattice of aspartic acid (asp) and valine (val) mostly revealed periodicities corresponding to bulk terminations, but a previously unreported molecular hexagonal lattice configuration was observed for both l-asp and l-val but not for d-asp or d-val. Atomic force microscopy can then be used to identify the different chiral forms of aspartic acid and valine crystals.

  5. Intersubunit communication in the dihydroorotase–aspartate transcarbamoylase complex of Aquifex aeolicus

    OpenAIRE

    Evans, Hedeel Guy; Fernando, Roshini; Vaishnav, Asmita; Kotichukkala, Mahalakshmi; Heyl, Deborah; Hachem, Fatme; Brunzelle, Joseph S.; Edwards, Brian FP; Evans, David R.

    2013-01-01

    Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solve...

  6. The Pathway of Product Release from the R State of Aspartate Transcarbamoylase

    OpenAIRE

    Mendes, Kimberly R.; Kantrowitz, Evan R.

    2010-01-01

    The pathway of product release from the R state of aspartate transcarbamoylase has been determined here by solving the crystal structure of Escherichia coli aspartate transcarbamoylase (ATCase) locked in the R-quaternary structure by specific introduction of disulfide bonds. ATCase displays ordered substrate binding and product release, remaining in the R state until substrates are exhausted. The structure reported here represents ATCase in the R state bound to the final product molecule, pho...

  7. Improved postprandial glycaemic control with insulin Aspart in type 2 diabetic patients treated with insulin

    DEFF Research Database (Denmark)

    Rosenfalck, A M; Thorsby, P; Kjems, L;

    2000-01-01

    The effect on postprandial blood glucose control of an immediately pre-meal injection of the rapid acting insulin analogue Aspart (IAsp) was compared with that of human insulin Actrapid injected immediately or 30 minutes before a test meal in insulin-treated type 2 diabetic patients with residual...... that the improved glucose control previously demonstrated with insulin Aspart compared to human insulin in healthy subjects and type 1 diabetic patients also applies to insulin-treated type 2 diabetic patients....

  8. Insulin Aspart in the Management of Diabetes Mellitus: 15 Years of Clinical Experience

    OpenAIRE

    Hermansen, Kjeld; Bohl, Mette; Schioldan, Anne Grethe

    2015-01-01

    Limiting excessive postprandial glucose excursions is an important component of good overall glycemic control in diabetes mellitus. Pharmacokinetic studies have shown that insulin aspart, which is structurally identical to regular human insulin except for the replacement of a single proline amino acid with an aspartic acid residue, has a more physiologic time–action profile (i.e., reaches a higher peak and reaches that peak sooner) than regular human insulin. As expected with this improved ph...

  9. A route to anionic hydrophilic films of copolymers of l-leucine, l-aspartic acid and l-aspartic acid esters

    NARCIS (Netherlands)

    Sederel, W.L.; Bantjes, A.; Feijen, J.

    1975-01-01

    A series of copolymers of l-leucine and β-benzyl-l-aspartate [Leu/Asp(OBz)] covering the range 30–70 mol % of l-leucine, was synthesized by the N-carboxyanhydride (NCA) method. The copolymers were characterized by elemental analysis, infra-red spectroscopy and viscometry. For all compositions high m

  10. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Rajasthan cohort of the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    Akhil Joshi

    2013-01-01

    Full Text Available Background: The A 1 chieve, a multicentric (28 countries, 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726 in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Rajasthan, India. Results: A total of 477 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 340, insulin detemir (n = 90, insulin aspart (n = 37, basal insulin plus insulin aspart (n = 7 and other insulin combinations (n = 2. At baseline glycaemic control was poor for both insulin naïve (mean HbA 1 c: 8.3% and insulin user (mean HbA 1 c: 8.4% groups. After 24 weeks of treatment, both the groups showed improvement in HbA 1 c (insulin naïve: −0.9%, insulin users: −1.2%. Major hypoglycaemic events decreased from 0.5 events/patient-year to 0.0 events/patient-year in insulin naïve group while no change from baseline (1.3 events/patients-year was observed for insulin users. SADRs were not reported in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  11. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    Science.gov (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  12. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    Science.gov (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  13. Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major γ-D-glutamyl-L-lysyl-endopeptidase.

    Directory of Open Access Journals (Sweden)

    Krzysztof Regulski

    Full Text Available Peptidoglycan (PG is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75 was identified as the major one. This protein is the homolog of p75 (Msp1 major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.

  14. L-(4-/sup 11/C)aspartic acid: enzymatic synthesis, myocardial uptake, and metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Barrio, J.R.; Egbert, J.E.; Henze, E.; Schelbert, H.R.; Baumgartner, F.J.

    1982-01-01

    Sterile, pyrogen-free L-(4-/sup 11/C)aspartic acid was prepared from /sup 11/CO/sub 2/ using phosphoenolpyruvate carboxylase and glutamic/oxaloacetic acid transaminase immobilized on Sepharose supports to determine if it is a useful indicator for in vivo, noninvasive determination of myocardial metabolism. An intracoronary bolus injection of L-(4-/sup 11/C)aspartic acid into dog myocardium showed a triexponential clearance curve with maximal production of /sup 11/CO/sub 2/ 100 s after injection. Inactivation of myocardial transaminase activity modified the tracer clearance and inhibited the production of /sup 11/CO/sub 2/. Positron-computed tomography imaging showed that the /sup 11/C activities retained in rhesus monkey myocardium are higher than those observed in dog heart after intravenous injection of L-(4-/sup 11/C)aspartic acid. These findings demonstrated the rapid incorporation of the carbon skeleton of L-aspartic acid into the tricarboxylic acid cycle after enzymatic transamination in myocardium and suggested that L-(4-/sup 11/C)aspartic acid could be of value for in vivo, noninvasive assessment of local myocardial metabolism.

  15. Interaction between L-aspartate and the brucite [Mg(OH)2]-water interface

    Science.gov (United States)

    Estrada, Charlene F.; Sverjensky, Dimitri A.; Pelletier, Manuel; Razafitianamaharavo, Angélina; Hazen, Robert M.

    2015-04-01

    The interaction of biomolecules at the mineral-water interface could have played a prominent role in the emergence of more complex organic species in life's origins. Serpentinite-hosted hydrothermal vents may have acted as a suitable environment for this process to occur, although little is known about biomolecule-mineral interactions in this system. We used batch adsorption experiments and surface complexation modeling to study the interaction of L-aspartate onto a thermodynamically stable product of serpentinization, brucite [Mg(OH)2], over a wide range of initial aspartate concentrations at four ionic strengths governed by [Mg2+] and [Ca2+]. We observed that up to 1.0 μmol of aspartate adsorbed per m2 of brucite at pH ∼ 10.2 and low Mg2+ concentrations (0.7 × 10-3 M), but surface adsorption decreased at high Mg2+ concentrations (5.8 × 10-3 M). At high Ca2+ concentrations (4.0 × 10-3 M), aspartate surface adsorption doubled (to 2.0 μmol m-2), with Ca2+ adsorption at 29.6 μmol m-2. We used the extended triple-layer model (ETLM) to construct a quantitative thermodynamic model of the adsorption data. We proposed three surface reactions involving the adsorption of aspartate (HAsp-) and/or Ca2+ onto brucite:

  16. Anti-N-methyl-D-aspartate receptor encephalitis presenting with acute psychosis in a preteenage girl: a case report

    OpenAIRE

    Maggina Paraskevi; Mavrikou Mersini; Karagianni Stavroula; Skevaki Chrysanthi L; Triantafyllidou Antigoni; Voudris Constantinos; Katsarou Eustathia; Stamogiannou Lela; Mastroyianni Sotiria

    2012-01-01

    Abstract Introduction Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is a rare, newly defined autoimmune clinical entity that presents with atypical clinical manifestations. Most patients with anti-N-methyl-D-aspartate receptor encephalitis develop a progressive illness from psychosis into a state of unresponsiveness, with catatonic features often associated with abnormal movements and autonomic instability. This is the first report of anti-N-methyl-D-aspartate receptor encephal...

  17. Differential Aspartate Usage Identifies a Subset of Cancer Cells Particularly Dependent on OGDH

    Directory of Open Access Journals (Sweden)

    Eric L. Allen

    2016-10-01

    Full Text Available Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

  18. Preparation and properties of poly(aspartic acid)-based hydrogel

    Energy Technology Data Exchange (ETDEWEB)

    Park, H.D. [Korea Institute of Science and Technology, Seoul (Korea, Republic of); Kim, J.H. [SungKyunKwan University, Suwon (Korea, Republic of); Kim, S.H.; Kim, Y.H. [Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    1999-03-01

    High molecular weight polysuccinimide (PSI), as a precursor of poly (aspartic acid), was prepared by thermal polycondensation of L-aspartic acid. The molecular weight was high when phosphoric acid was used as a catalyst, and the ratio to monomer was 0.75 : 1(phosphoric acid : L-aspartic acid). Attempted solution polymerization in various sulfolane/mesitylene mixtures gave only low molecular weight polymers. By the post polymerization of PSI using DCC as a condensing reagent, the molecular weight of PSI could be increased to some extent. Hydrogels was prepared by crosslinking reaction of PSI with diamine, followed by hydrolysis with NaOH either in water or in DMF solution. As high as 104 g water/g-polymer absorption could be obtained from the hydrogel prepared with 3 mol % of hexamethylenediamine. 13 refs., 7 figs., 1 tab.

  19. Aspartate embedding depth affects pHLIP's insertion pKa.

    Science.gov (United States)

    Fendos, Justin; Barrera, Francisco N; Engelman, Donald M

    2013-07-01

    We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  20. Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate

    Energy Technology Data Exchange (ETDEWEB)

    Mise, Takeshi; Matsunami, Hideyuki; Samatey, Fadel A.; Maruyama, Ichiro N., E-mail: ichi@oist.jp [Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami, Okinawa 904-0495 (Japan)

    2014-08-27

    The periplasmic domain of the E. coli aspartate receptor Tar was cloned, expressed, purified and crystallized with and without bound ligand. The crystals obtained diffracted to resolutions of 1.58 and 1.95 Å, respectively. The cell-surface receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni{sup 2+}. To understand the molecular mechanisms underlying the induction of Tar activity by its ligands, the Escherichia coli Tar periplasmic domain with and without bound aspartate (Asp-Tar and apo-Tar, respectively) were each crystallized in two different forms. Using ammonium sulfate as a precipitant, crystals of apo-Tar1 and Asp-Tar1 were grown and diffracted to resolutions of 2.10 and 2.40 Å, respectively. Alternatively, using sodium chloride as a precipitant, crystals of apo-Tar2 and Asp-Tar2 were grown and diffracted to resolutions of 1.95 and 1.58 Å, respectively. Crystals of apo-Tar1 and Asp-Tar1 adopted space group P4{sub 1}2{sub 1}2, while those of apo-Tar2 and Asp-Tar2 adopted space groups P2{sub 1}2{sub 1}2{sub 1} and C2, respectively.

  1. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Qatar cohort of the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    Mohamed Hasan Daghash

    2013-01-01

    Full Text Available Background: The A 1 chieve, a multicentric (28 countries, 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726 in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Qatar. Results: A total of 91 patients were enrolled in the study. Two insulin analogue regimens were used in the study. Study patients had started on or were switched to biphasic insulin aspart (n = 88, insulin detemir (n = 2, and other insulin combinations (n = 1. At baseline glycaemic control was poor for both insulin naïve (mean HbA 1 c: 10.9% and insulin users (mean HbA 1 c: 9.1% groups. After 24 weeks of treatment, all the study groups showed improvement in HbA 1 c (insulin naïve: −1.8%, insulin users: −1.3%. Major hypoglycaemia did not occur in the study patients. SADRs were reported in 1.4% of insulin users. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  2. Toll-like receptor 5 (TLR5), IL-1β secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

    Science.gov (United States)

    Descamps, Delphyne; Le Gars, Mathieu; Balloy, Viviane; Barbier, Diane; Maschalidi, Sophia; Tohme, Mira; Chignard, Michel; Ramphal, Reuben; Manoury, Bénédicte; Sallenave, Jean-Michel

    2012-01-31

    A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation. PMID:22307620

  3. Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

    Science.gov (United States)

    Valdés-Rodríguez, Silvia; Galván-Ramírez, Juan Pablo; Guerrero-Rangel, Armando; Cedro-Tanda, Alberto

    2015-01-01

    In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity. PMID:25345487

  4. Aza-peptidyl Michael acceptor and epoxide inhibitors--potent and selective inhibitors of Schistosoma mansoni and Ixodes ricinus legumains (asparaginyl endopeptidases).

    Science.gov (United States)

    Ovat, Asli; Muindi, Fanuel; Fagan, Crystal; Brouner, Michelle; Hansell, Elizabeth; Dvorák, Jan; Sojka, Daniel; Kopácek, Petr; McKerrow, James H; Caffrey, Conor R; Powers, James C

    2009-11-26

    Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.

  5. Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

    Science.gov (United States)

    Agarwal, Vaibhav; Sroka, Magdalena; Fulde, Marcus; Bergmann, Simone; Riesbeck, Kristian; Blom, Anna M

    2014-05-30

    The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

  6. Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

    Science.gov (United States)

    Valdés-Rodríguez, Silvia; Galván-Ramírez, Juan Pablo; Guerrero-Rangel, Armando; Cedro-Tanda, Alberto

    2015-01-01

    In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.

  7. N-Hydroxypyrazolyl glycine derivatives as selective N-methyl-D-aspartic acid receptor ligands

    DEFF Research Database (Denmark)

    Clausen, Rasmus Prætorius; Christensen, Caspar; Hansen, Kasper Bø;

    2008-01-01

    glycine (NHP5G) derivatives are selectively recognized by N-methyl- d-aspartic acid (NMDA) receptors and that the ( R)-enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual subtypes among the NMDA receptors, providing new pharmacological tools. For......A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl...

  8. Proton transfer pathways in an aspartate-water cluster sampled by a network of discrete states

    Science.gov (United States)

    Reidelbach, Marco; Betz, Fridtjof; Mäusle, Raquel Maya; Imhof, Petra

    2016-08-01

    Proton transfer reactions are complex transitions due to the size and flexibility of the hydrogen-bonded networks along which the protons may "hop". The combination of molecular dynamics based sampling of water positions and orientations with direct sampling of proton positions is an efficient way to capture the interplay of these degrees of freedom in a transition network. The energetically most favourable pathway in the proton transfer network computed for an aspartate-water cluster shows the pre-orientation of water molecules and aspartate side chains to be a pre-requisite for the subsequent concerted proton transfer to the product state.

  9. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, Henrik; Galili, G; Knudsen, S;

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the...

  10. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    Science.gov (United States)

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  11. Aspartic acid in the hippocampus:a biomarker for postoperative cognitive dysfunction

    Institute of Scientific and Technical Information of China (English)

    Rong Hu; Dong Huang; Jianbin Tong; Qin Liao; Zhonghua Hu; Wen Ouyang

    2014-01-01

    This study established an aged rat model of cognitive dysfunction using anesthesia with 2%iso-lfurane and 80%oxygen for 2 hours. Twenty-four hours later, Y-maze test results showed that isoflurane significantly impaired cognitive function in aged rats. Gas chromatography-mass spectrometry results showed that isolfurane also signiifcantly increased the levels of N,N-diethy-lacetamide, n-ethylacetamide, aspartic acid, malic acid and arabinonic acid in the hippocampus of isolfurane-treated rats. Moreover, aspartic acid, N,N-diethylacetamide, n-ethylacetamide and malic acid concentration was positively correlated with the degree of cognitive dysfunction in the isolfurane-treated rats. It is evident that hippocampal metabolite changes are involved in the formation of cognitive dysfunction after isoflurane anesthesia. To further verify these results, this study cultured hippocampal neurons in vitro, which were then treated with aspartic acid (100 µmol/L). Results suggested that aspartic acid concentration in the hippocampus may be a biomarker for predicting the occurrence and disease progress of cognitive dysfunction.

  12. Leukotriene receptor antagonist ONO-1078 attenuate N-metyl-D-aspartate - mediated neurotoxicity in rats

    Institute of Scientific and Technical Information of China (English)

    ZHANGLi-Hui; WEIEr-Qing

    2004-01-01

    AIM: To investigate the possible effect of ONO-1078 t pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy) benzoyl-amono]-2- (tetrazol-5-yl)-4H-1-benzopyran hemihydrate],a potent leukotriene receptor antagonist,on N-metyl-D-aspartate (NMDA)-mediated neurotoxicity in rats. METHODS: Brain injury was induced by NM-

  13. An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

    NARCIS (Netherlands)

    Have, ten A.; Dekkers, E.; Kay, J.; Phylip, L.H.; Kan, van J.A.L.

    2004-01-01

    Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medi

  14. Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

    DEFF Research Database (Denmark)

    Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

    2009-01-01

    that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

  15. Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation

    DEFF Research Database (Denmark)

    Avanti, Christina; Oktaviani, Nur Alia; Hinrichs, Wouther L.J.;

    2013-01-01

    is improved by the addition of divalent metal ions (unpublished results). The stabilizing effect of Zn2+ was by far superior compared to that of Mg2+. In addition, it was found that stabilization correlated well with the ability of the divalent metal ions to interact with oxytocin in aspartate buffer...

  16. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture

    DEFF Research Database (Denmark)

    Balázs, R; Jørgensen, Ole Steen; Hack, N

    1988-01-01

    and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved...

  17. Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh

    NARCIS (Netherlands)

    Hänelt, Inga; Jensen, Sonja; Wunnicke, Dorith; Slotboom, Dirk Jan

    2015-01-01

    GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters (EAATs) that take up the neurotransmitter glutamate. Each protomer in GltPh consis

  18. The formation of oxytocin dimers is suppressed by the zinc-aspartate-oxytocin complex

    NARCIS (Netherlands)

    Avanti, Christina; Hinrichs, Wouter L. J.; Casini, Angela; Eissens, Anko C.; van Dam, Annigje; Kedrov, Alexej; Driessen, Arnold J. M.; Frijlink, Henderik W.; Permentier, Hjalmar P.

    2013-01-01

    The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg2+, and Zn2+) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liquid chromatography and high-performanc

  19. Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation

    NARCIS (Netherlands)

    Avanti, Christina; Oktaviani, Nur Alia; Hinrichs, Wouter L J; Frijlink, Henderik W; Mulder, Frans A A

    2013-01-01

    Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under tropical conditions is problematic. In a previous study, we have found that the stability of oxytocin in aspartate buffered formulation

  20. Searsia species with affinity to the N-methyl-d-aspartic acid (NMDA) receptor

    DEFF Research Database (Denmark)

    Jäger, Anna; Knap, D.M.; Nielsen, Birgitte;

    2012-01-01

    Species of Searsia are used in traditional medicine to treat epilepsy. Previous studies on S. dentata and S. pyroides have shown that this is likely mediated via the N-methyl-d-aspartic acid (NMDA) receptor. Ethanolic extracts of leaves of six Searsia species were tested in a binding assay for...... accessible Searsia species can be used in traditional medicine....

  1. Hypoglycemia in type 1 diabetic pregnancy: role of preconception insulin aspart treatment in a randomized study

    DEFF Research Database (Denmark)

    Heller, Simon; Damm, Peter; Mersebach, Henriette;

    2010-01-01

    OBJECTIVE A recent randomized trial compared prandial insulin aspart (IAsp) with human insulin in type 1 diabetic pregnancy. The aim of this exploratory analysis was to investigate the incidence of severe hypoglycemia during pregnancy and compare women enrolled preconception with women enrolled...

  2. N-methyl-D-aspartate promotes the survival of cerebellar granule cells: pharmacological characterization

    DEFF Research Database (Denmark)

    Balázs, R; Hack, N; Jørgensen, Ole Steen;

    1989-01-01

    The survival of cerebellar granule cells in culture is promoted by chronic exposure to N-methyl-D-aspartate (NMDA). The effect is due to the stimulation of 'conventional' NMDA receptor-ionophore complex: it is concentration dependent, voltage dependent and blocked by the selective antagonists D-2...

  3. AspC-mediated aspartate metabolism coordinates the Escherichia coli cell cycle.

    Directory of Open Access Journals (Sweden)

    Feng Liu

    Full Text Available The fast-growing bacterial cell cycle consists of at least two independent cycles of chromosome replication and cell division. To ensure proper cell cycles and viability, chromosome replication and cell division must be coordinated. It has been suggested that metabolism could affect the Escherichia coli cell cycle, but the idea is still lacking solid evidences.We found that absence of AspC, an aminotransferase that catalyzes synthesis of aspartate, led to generation of small cells with less origins and slow growth. In contrast, excess AspC was found to exert the opposite effect. Further analysis showed that AspC-mediated aspartate metabolism had a specific effect in the cell cycle, as only extra aspartate of the 20 amino acids triggered production of bigger cells with more origins per cell and faster growth. The amount of DnaA protein per cell was found to be changed in response to the availability of AspC. Depletion of (pppGpp by ΔrelAΔspoT led to a slight delay in initiation of replication, but did not change the replication pattern found in the ΔaspC mutant.The results suggest that AspC-mediated metabolism of aspartate coordinates the E. coli cell cycle through altering the amount of the initiator protein DnaA per cell and the division signal UDP-glucose. Furthermore, AspC sequence conservation suggests similar functions in other organisms.

  4. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander (Guelph); (Kyoto); (NCI)

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  5. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    Energy Technology Data Exchange (ETDEWEB)

    Onstott, T. C. [Princeton University; Aubrey, A.D. [Jet Propulsion Laboratory, Pasadena, CA; Kieft, T L [New Mexico Institute of Mining and Technology; Silver, B J [Jet Propulsion Laboratory, Pasadena, CA; Phelps, Tommy Joe [ORNL; Van Heerden, E. [University of the Free State; Opperman, D. J. [University of the Free State; Bada, J L. [Geosciences Research Division, Scripps Instition of Oceanography, Univesity of California San Diego,

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 C and 1 2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  6. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    Science.gov (United States)

    Onstott, T C; Magnabosco, C; Aubrey, A D; Burton, A S; Dworkin, J P; Elsila, J E; Grunsfeld, S; Cao, B H; Hein, J E; Glavin, D P; Kieft, T L; Silver, B J; Phelps, T J; van Heerden, E; Opperman, D J; Bada, J L

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples. PMID:24289240

  7. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    Science.gov (United States)

    Onstott, T C; Magnabosco, C; Aubrey, A D; Burton, A S; Dworkin, J P; Elsila, J E; Grunsfeld, S; Cao, B H; Hein, J E; Glavin, D P; Kieft, T L; Silver, B J; Phelps, T J; van Heerden, E; Opperman, D J; Bada, J L

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  8. Does Aspartic Acid Racemization Constrain the Depth Limit of the Subsurface Biosphere?

    Science.gov (United States)

    Onstott, T C.; Magnabosco, C.; Aubrey, A. D.; Burton, A. S.; Dworkin, J. P.; Elsila, J. E.; Grunsfeld, S.; Cao, B. H.; Hein, J. E.; Glavin, D. P.; Kieft, T. L.; Silver, B. J.; Phelps, T. J.; Heerden, E. Van; Opperman, D. J.; Bada, J. L.

    2013-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of approximately 89 years for 1 km depth and 27 C and 1-2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  9. Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

    Science.gov (United States)

    Cunin, R; Rani, C S; Van Vliet, F; Wild, J R; Wales, M

    1999-12-17

    The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified. PMID:10600394

  10. Abundance of cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) is regulated by different stimuli from prey through jasmonates.

    Science.gov (United States)

    Libiaková, Michaela; Floková, Kristýna; Novák, Ondřej; Slováková, L'udmila; Pavlovič, Andrej

    2014-01-01

    The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH4Cl, KH2PO4, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed. PMID:25153528

  11. Neurotensin high affinity binding sites and endopeptidase 24. 11 are present respectively in the meningothelial and in the fibroblastic components of human meningiomas

    Energy Technology Data Exchange (ETDEWEB)

    Mailleux, P.; Przedborski, S.; Beaumont, A.; Verslijpe, M.; Depierreux, M.; Levivier, M.; Kitabgi, P.; Roques, B.P.; Vanderhaeghen, J.J. (Brugmann Hospital, Bruxelles (Belgium))

    1990-11-01

    The presence of neurotensin receptors and endopeptidase 24.11 (E-24.11) in 16 human meningioma specimens, obtained at surgery, was assessed by measuring the binding of {sup 125}I-(tyrosyl3)neurotensin(1-13) ({sup 125}I-NT) and the inhibitor {sup 3}H-N(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-(oxopropyl)glycine ({sup 3}H-HACBO-Gly), for the receptor and enzyme, respectively. E-24.11 activity was also measured. Autoradiography, on the 16 meningiomas, showed that specific {sup 125}I-NT labeling (nonspecific labeling was assessed in the presence of excess NT) was exclusively located in the meningothelial regions. In contrast, specific {sup 3}H-HACBO-Gly labeling (nonspecific labeling was assessed in the presence of an excess of the E-24.11 inhibitor thiorphan) was exclusively found in fibroblastic regions. No specific labeling of either ligand was found on collagen or blood vessels. In vitro binding assays were performed on membranes of 10 of the 16 meningiomas. In the 4 meningiomas rich in meningothelial cells, {sup 125}I-NT specifically bound to one population of sites with Bmax ranging from 57 to 405 fmol/mg protein and Kd around 0.3 nM. These sites share common properties with the brain NT receptor, since the carboxy terminal acetyl NT(8-13) fragment bound to the same sites but with a higher affinity. The carboxy terminal analogue of NT, neuromedin N, also bound to the same sites with a 10-fold lower affinity and the sites were bradykinin and levocabastine insensitive. In the 4 meningiomas rich in fibroblastic cells, {sup 3}H-HACBO-Gly specifically bound to one population of sites with Bmax ranging from 251 to 739 fmol/mg protein and Kd around 2.8 nM.

  12. Hypoxia increases membrane metallo-endopeptidase expression in a novel lung cancer ex vivo model – role of tumor stroma cells

    International Nuclear Information System (INIS)

    Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo. Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays. Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase (MME, neprilysin, CD10) expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets. The novel ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced

  13. Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

    OpenAIRE

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; WANG, XICHENG; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

    2007-01-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter cla...

  14. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    Science.gov (United States)

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. PMID:27438592

  15. Effects of Glutamate and Aspartate on Serum Antioxidative Enzyme, Sex Hormones, and Genital Inflammation in Boars Challenged with Hydrogen Peroxide

    Directory of Open Access Journals (Sweden)

    Hengjia Ni

    2016-01-01

    Full Text Available Background. Oxidative stress is associated with infertility. This study was conducted to determine the effects of glutamate and aspartate on serum antioxidative enzymes, sex hormones, and genital inflammation in boars suffering from oxidative stress. Methods. Boars were randomly divided into 4 groups: the nonchallenged control (CON and H2O2-challenged control (BD groups were fed a basal diet supplemented with 2% alanine; the other two groups were fed the basal diet supplemented with 2% glutamate (GLU or 2% aspartate (ASP. The BD, GLU, and ASP groups were injected with hydrogen peroxide (H2O2 on day 15. The CON group was injected with 0.9% sodium chloride solution on the same day. Results. Dietary aspartate decreased the malondialdehyde (MDA level in serum (P<0.05 compared with the BD group. Additionally, aspartate maintained serum luteinizing hormone (LH at a relatively stable level. Moreover, glutamate and aspartate increased transforming growth factor-β1 (TGF-β1 and interleukin-10 (IL-10 levels in the epididymis and testis (P<0.05 compared with the BD group. Conclusion. Both glutamate and aspartate promoted genital mRNA expressions of anti-inflammatory factors after oxidative stress. Aspartate more effectively decreased serum MDA and prevented fluctuations in serum sex hormones after H2O2 challenge than did glutamate.

  16. Solvent-Free Polymerization of L-Aspartic Acid in the Presence of D-Sorbitol to Obtain Water Soluble or Network Copolymers

    Science.gov (United States)

    L-aspartic acid was thermally polymerized in the presence of D-sorbitol with the goal of synthesizing new, higher molecular weight water soluble and absorbent copolymers. No reaction occurred when aspartic acid alone was heated at 170 or 200 degrees C. In contrast, heating sorbitol and aspartic ac...

  17. The Reovirus Sigmal Aspartic Acid Sandwich: A Trimerization Motif Poised for Conformational Change

    Energy Technology Data Exchange (ETDEWEB)

    Schelling,P.; Guglielml, K.; Kirchner, E.; Paetzold, b.; Dermody, T.; Stehle, T.

    2007-01-01

    Reovirus attachment protein {sigma}1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The {sigma}1 protein is a filamentous, trimeric molecule with a globular {beta}-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the {sigma}1 subunit interface. A 1.75 {angstrom} structure of the {sigma}1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the {sigma}1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.

  18. The reovirus sigma1 aspartic acid sandwich: a trimerization motif poised for conformational change.

    Science.gov (United States)

    Schelling, Pierre; Guglielmi, Kristen M; Kirchner, Eva; Paetzold, Bernhard; Dermody, Terence S; Stehle, Thilo

    2007-04-13

    Reovirus attachment protein sigma1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The sigma1 protein is a filamentous, trimeric molecule with a globular beta-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the sigma1 subunit interface. A 1.75-A structure of the sigma1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the sigma1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.

  19. Membrane topology of the electrogenic aspartate-alanine antiporter AspT of Tetragenococcus halophilus.

    Science.gov (United States)

    Nanatani, Kei; Ohonishi, Fumito; Yoneyama, Hiroshi; Nakajima, Tasuku; Abe, Keietsu

    2005-03-01

    AspT is an electrogenic aspartate:alanine exchange protein that represents the vectorial component of a proton-motive metabolic cycle found in some strains of Tetragenococcus halophilus. AspT is the sole member of a new family, the Aspartate: Alanine Exchanger (AAE) family, in secondary transporters, according to the computational classification proposed by Saier et al. (http://www.biology.ucsd.edu/~msaier/transport/). We analyzed the topology of AspT biochemically, by using fusion methods in combination with alkaline phosphatase or beta-lactamase. These results suggested that AspT has a unique topology; 8 TMS, a large cytoplasmic loop (183 amino acids) between TMS5 and TMS6, and N- and C-termini that both face the periplasm. These results demonstrated a unique 2D-structure of AspT as the novel AAE family. PMID:15670744

  20. An easy method for diagnosing macro-aspartate aminotransferase: a case series.

    Science.gov (United States)

    Beşer, Omer Faruk; Laçinel, Sibel; Gülcü, Didem; Kutlu, Tufan; Cullu Çokuğraş, Fügen; Erkan, Tülay

    2014-10-01

    Macro-aspartate transaminase (macro-AST) must be considered when the aspartate transaminase (AST) level is chronically high without any liver, cardiac, or muscle disease. Many specialized laboratory techniques have been recommended for diagnosing macro-AST, including the polyethylene glycol immune precipitate technique, which is simple. This study presents a considerably easier method based on the studies of Davidson and Watson and Castiella et al. Our method is based on the decrease in the plasma AST level after storage of the macroenzyme at 2-8 °C for 5 days, and has the advantages of low cost, reliability, and practicality at any health center. In our eight cases of macro-AST, the AST activity at day 6 had decreased by more than 50% from day 1. This method is practical for primary healthcare facilities because of its easy application and accurate results, and obviated the need for unnecessary tests after diagnosis.

  1. Seed-specific aspartic proteinase FeAP12 from buckwheat (Fagopyrum esculentum Moench

    Directory of Open Access Journals (Sweden)

    Timotijević Gordana S.

    2010-01-01

    Full Text Available Aspartic proteinase gene (FeAP12 has been isolated from the cDNA library of developing buckwheat seeds. Analysis of its deduced amino acid sequence showed that it resembled the structure and shared high homology with typical plant aspartic proteinases (AP characterized by the presence of a plant-specific insert (PSI, unique among APs. It was shown that FeAP12 mRNA was not present in the leaves, roots, steam and flowers, but was seed-specifically expressed. Moreover, the highest levels of FeAP12 expression were observed in the early stages of seed development, therefore suggesting its potential role in nucellar degradation.

  2. Pragmatic use of insulin degludec/insulin aspart co-formulation: A multinational consensus statement

    Science.gov (United States)

    Kalra, Sanjay; Latif, Zafar A.; Comlekci, Abdurrahman; Galvez, Guillermo Gonzalez; Malik, Rached; Pathan, Md Faruque; Kumar, Ajay

    2016-01-01

    Insulin degludec/insulin aspart (IDegAsp) is a modern coformulation of ultra-long-acting basal insulin degludec, with rapid-acting insulin aspart. IDegAsp provides effective, safe, well-tolerated glycemic control, with a low risk of hypoglycemia while allowing flexibility in meal patterns and timing of administration. This consensus statement describes a pragmatic framework to identify patients who may benefit from IDegAsp therapy. It highlights the utility of IDegAsp in type 2 diabetic patients who are insulin-naive, suboptimally controlled on basal or premixed insulin, or dissatisfied with basal–bolus regimens. It also describes potential IDegAsp usage in type 1 diabetic patients. PMID:27366723

  3. Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Arévalo-Rodríguez, M; Calderón, I L; Holmberg, S

    1999-01-01

    . The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved...... inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP...

  4. A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids.

    OpenAIRE

    El-Azaz, Jorge; Cánovas, Francisco M.; de la Torre, Fernando; Ávila, Concepción

    2014-01-01

    A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids. Fernando de la Torre, Jorge El-Azaz, Concepción Ávila, Francisco M. Cánovas Departamento de Biología Molecular y Bioquímica. Universidad de Málaga. Bifunctional aspartate/prephenate aminotransferases (AAT/PAT) are plastid-located enzymes encoded by a single locus in all reported plants, which develop two different enzymatic activities: aspartate aminotransferas...

  5. Absorption and utilization of organic matter by the strict autotroph, Thiobacillus thiooxidans, with special reference to aspartic acid.

    Science.gov (United States)

    Butler, R G; Umbreit, W W

    1966-02-01

    Butler, Richard G. (Rutgers, The State University, New Brunswick, N.J.), and Wayne W. Umbreit. Absorption and utilization of organic matter by the strict autotroph, Thiobacillus thiooxidans, with special reference to aspartic acid. J. Bacteriol. 91:661-666. 1966.-The strictly autotrophic bacterium, Thiobacillus thiooxidans, can be shown to assimilate a variety of organic materials. Aspartic acid can be assimilated into protein and can be converted into CO(2), but even in the presence of sulfur it cannot serve as the sole source of carbon for growth. The reason appears to be that aspartic acid is converted into inhibitory materials.

  6. N-(Fluoren-9-ylmethoxycarbonyl-l-aspartic acid 4-tert-butyl ester

    Directory of Open Access Journals (Sweden)

    Kazuhiko Yamada

    2009-11-01

    Full Text Available The bond distances and bond angles of the title compound, C23H25NO6, are consistent with values typically found for fluoren-9-ylmethoxycarbonyl-protected amino acids. The conformations of the backbone and the side chain are slightly different from those of l-aspartic acid. The crystal structure exhibits two intermolecular hydrogen bonds, forming a two-dimensional sheet structure parallel to the ab plane.

  7. Anti-N-methyl-D-aspartate-receptor encephalitis: diagnosis, optimal management, and challenges

    OpenAIRE

    Mann, Andrea

    2014-01-01

    Andrea P Mann,1 Elena Grebenciucova,2 Rimas V Lukas21Department of Psychiatry and Behavioral Neuroscience, 2Department of Neurology, University of Chicago, Chicago, IL, USAObjective: Anti-N-methyl-D-aspartate-receptor (NMDA-R) encephalitis is a new autoimmune disorder, often paraneoplastic in nature, presenting with complex neuropsychiatric symptoms. Diagnosed serologically, this disorder is often responsive to immunosuppressant treatment. The objective of this review is to educate clinicians...

  8. Selective Vulnerabilities of N-methyl-D-aspartate (NMDA) Receptors During Brain Aging

    OpenAIRE

    Magnusson, Kathy R.; Brenna L Brim; Das, Siba R.

    2010-01-01

    N-methyl-D-aspartate (NMDA) receptors are present in high density within the cerebral cortex and hippocampus and play an important role in learning and memory. NMDA receptors are negatively affected by aging, but these effects are not uniform in many different ways. This review discusses the selective age-related vulnerabilities of different binding sites of the NMDA receptor complex, different subunits that comprise the complex, and the expression and functions of the receptor within differe...

  9. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    OpenAIRE

    Torruella, M; Gordon, K; Hohn, T

    1989-01-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivit...

  10. Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis.

    Science.gov (United States)

    Rabinovich, Shiran; Adler, Lital; Yizhak, Keren; Sarver, Alona; Silberman, Alon; Agron, Shani; Stettner, Noa; Sun, Qin; Brandis, Alexander; Helbling, Daniel; Korman, Stanley; Itzkovitz, Shalev; Dimmock, David; Ulitsky, Igor; Nagamani, Sandesh C S; Ruppin, Eytan; Erez, Ayelet

    2015-11-19

    Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.

  11. Insulin degludec and insulin aspart: novel insulins for the management of diabetes mellitus

    OpenAIRE

    Atkin, Stephen; Javed, Zeeshan; Fulcher, Gregory

    2015-01-01

    Patients with type 2 diabetes mellitus require insulin as disease progresses to attain or maintain glycaemic targets. Basal insulin is commonly prescribed initially, alone or with one or more rapid-acting prandial insulin doses, to limit mealtime glucose excursions (a basal–bolus regimen). Both patients and physicians must balance the advantages of improved glycaemic control with the risk of hypoglycaemia and increasing regimen complexity. The rapid-acting insulin analogues (insulin aspart, i...

  12. Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris

    OpenAIRE

    Gama Salgado, Jose Antonio; Kangwa, Martin; Fernandez-Lahore, Marcelo

    2013-01-01

    Background Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme. Results Th...

  13. Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bhaumik, Prasenjit; Xiao, Huogen; Parr, Charity L.; Kiso, Yoshiaki; Gustchina, Alla; Yada, Rickey Y.; Wlodawer, Alexander; (Guelph); (Kyoto); (NCI)

    2009-08-07

    The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5-, 3.3-, and 3.05-{angstrom} resolutions, respectively. In the apoenzyme crystals, HAP forms a tight dimer not seen previously in any aspartic protease. The interactions between the monomers affect the conformation of two flexible loops, the functionally important 'flap' (residues 70-83) and its structural equivalent in the C-terminal domain (residues 238-245), as well as the orientation of helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different from that in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared with the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

  14. Anti-N-Methyl-D-Aspartate Receptor Encephalitis in Korea: Clinical Features, Treatment, and Outcome

    OpenAIRE

    Lim, Jung-Ah; Lee, Soon-Tae; Jung, Keun-Hwa; Kim, Soyun; Shin, Jung-Won; Moon, Jangsup; Byun, Jung-Ick; Kim, Tae-Joon; Shin, Yong-Won; Lee, Keon-Joo; Kim, Young-Su; Park, Kyung-Il; Lee, Sang Kun; Chu, Kon

    2014-01-01

    Background and Purpose Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is the most common type of autoimmune synaptic encephalitis and it often responds to treatment. We analyzed the clinical characteristics of anti-NMDAR encephalitis in Korea. Methods Serum and/or cerebrospinal fluid (CSF) of adult patients (aged ≥18 years) with encephalitis of undetermined cause were screened for anti-NMDAR antibodies using a cell-based indirect immunofluorescence assay. The patients came from ...

  15. Age estimation in forensic sciences: Application of combined aspartic acid racemization and radiocarbon analysis

    Energy Technology Data Exchange (ETDEWEB)

    Alkass, K; Buchholz, B A; Ohtani, S; Yamamoto, T; Druid, H; Spalding, S L

    2009-11-02

    Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster, since the age at death, birth date and year of death, as well as gender, can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization has shown reproducible and more precise results. In this paper we analyze teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that above-ground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ({sup 14}C) which have been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel and ten of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R2=0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 0.6 {+-} 04 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 {+-} 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

  16. Diversity in anti-N-methyl-D-aspartate receptor encephalitis: case-based evidence

    OpenAIRE

    Pinho, J; Rocha, J.; Rodrigues, M.; Pereira, J.; Maré, R; Ferreira, C; Lourenço, E; Beleza, P

    2012-01-01

    Antibodies against N-methyl-D-aspartate receptor (NMDAR) are identified in the form of immune-mediated encephalitis in which typical manifestations include neuropsychiatric symptoms, seizures, abnormal movements, dysautonomia and hypoventilation. The authors report two cases of anti-NMDAR encephalitis with different presentations and patterns of progression. The first patient presented with status epilepticus and later developed psychosis, pyramidal signs and diffuse encephalopathy. The secon...

  17. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

  18. Intersubunit communication in the dihydroorotase-aspartate transcarbamoylase complex of Aquifex aeolicus.

    Science.gov (United States)

    Evans, Hedeel Guy; Fernando, Roshini; Vaishnav, Asmita; Kotichukkala, Mahalakshmi; Heyl, Deborah; Hachem, Fatme; Brunzelle, Joseph S; Edwards, Brian F P; Evans, David R

    2014-01-01

    Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K(i) = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes. PMID:24353170

  19. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle. PMID:198130

  20. Occurrence of the malate-aspartate shuttle in various tumor types.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1976-04-01

    The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm. PMID:177206

  1. The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

    Science.gov (United States)

    D'Abrosca, Gianluca; Russo, Luigi; Palmieri, Maddalena; Baglivo, Ilaria; Netti, Fortuna; de Paola, Ivan; Zaccaro, Laura; Farina, Biancamaria; Iacovino, Rosa; Pedone, Paolo Vincenzo; Isernia, Carla; Fattorusso, Roberto; Malgieri, Gaetano

    2016-08-01

    The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties. PMID:27238756

  2. N-Methyl-D-aspartic Acid (NMDA in the nervous system of the amphioxus Branchiostoma lanceolatum

    Directory of Open Access Journals (Sweden)

    Garcia-Fernàndez Jordi

    2007-12-01

    Full Text Available Abstract Background NMDA (N-methyl-D-aspartic acid is a widely known agonist for a class of glutamate receptors, the NMDA type. Synthetic NMDA elicits very strong activity for the induction of hypothalamic factors and hypophyseal hormones in mammals. Moreover, endogenous NMDA has been found in rat, where it has a role in the induction of GnRH (Gonadotropin Releasing Hormone in the hypothalamus, and of LH (Luteinizing Hormone and PRL (Prolactin in the pituitary gland. Results In this study we show evidence for the occurrence of endogenous NMDA in the amphioxus Branchiostoma lanceolatum. A relatively high concentration of NMDA occurs in the nervous system of this species (3.08 ± 0.37 nmol/g tissue in the nerve cord and 10.52 ± 1.41 nmol/g tissue in the cephalic vesicle. As in rat, in amphioxus NMDA is also biosynthesized from D-aspartic acid (D-Asp by a NMDA synthase (also called D-aspartate methyl transferase. Conclusion Given the simplicity of the amphioxus nervous and endocrine systems compared to mammalian, the discovery of NMDA in this protochordate is important to gain insights into the role of endogenous NMDA in the nervous and endocrine systems of metazoans and particularly in the chordate lineage.

  3. N-Methyl-D-Aspartate Receptor Signaling and Function in Cardiovascular Tissues.

    Science.gov (United States)

    McGee, Marie A; Abdel-Rahman, Abdel A

    2016-08-01

    Excellent reviews on central N-methyl-D-aspartate receptor (NMDAR) signaling and function in cardiovascular regulating neuronal pools have been reported. However, much less attention has been given to NMDAR function in peripheral tissues, particularly the heart and vasculature, although a very recent review discusses such function in the kidney. In this short review, we discuss the NMDAR expression and complexity of its function in cardiovascular tissues. In conscious (contrary to anesthetized) rats, activation of the peripheral NMDAR triggers cardiovascular oxidative stress through the PI3K-ERK1/2-NO signaling pathway, which ultimately leads to elevation in blood pressure. Evidence also implicates Ca release, in the peripheral NMDAR-mediated pressor response. Despite evidence of circulating potent ligands (eg, D-aspartate and L-aspartate, L-homocysteic acid, and quinolinic acid) and also their coagonist (eg, glycine or D-serine), the physiological role of peripheral cardiovascular NMDAR remains elusive. Nonetheless, the cardiovascular relevance of the peripheral NMDAR might become apparent when its signaling is altered by drugs, such as alcohol, which interact with the NMDAR or its downstream signaling mechanisms. PMID:27046337

  4. Potent new small-molecule inhibitor of botulinum neurotoxin serotype A endopeptidase developed by synthesis-based computer-aided molecular design.

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    Full Text Available Botulinum neurotoxin serotype A (BoNTA causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i (app value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50 value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2 for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD. This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight

  5. Gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase as markers of alcohol consumption in out-patient alcoholics

    DEFF Research Database (Denmark)

    Gluud, C; Andersen, I; Dietrichson, O;

    1981-01-01

    Serum activity of gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase were determined in 316 patients attending an out-patients clinic for treatment of alcoholism. The activity of gamma-glutamyltransferase was raised in 34% and that of aspartate aminotransferase...... and alkaline phosphatase in 18% and 7%. Neither the activity of gamma-glutamyltransferase, aspartate aminotransferase nor alkaline phosphatase showed any significant (P greater than 0.05) correlation with the history of alcohol consumption. The activities of gamma-glutamyltransferase and aspartate...... aminotransferase were raised significantly more often in patients with recent alcohol consumption than in patients who had abstained for more than 9 days. The concentration of alkaline phosphatase was not significantly (P greater than 0.05) different in these groups. The predictive value of raised and normal...

  6. The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate.

    OpenAIRE

    Anis, N. A.; Berry, S. C.; Burton, N. R.; Lodge, D.

    1983-01-01

    The interaction of two dissociative anaesthetics, ketamine and phencyclidine, with the responses of spinal neurones to the electrophoretic administration of amino acids and acetylcholine was studied in decerebrate or pentobarbitone-anaesthetized cats and rats. Both ketamine and phencyclidine selectively blocked excitation by N-methyl-aspartate (NMA) with little effect on excitation by quisqualate and kainate. Ketamine reduced responses to L-aspartate somewhat more than those of L-glutamate; t...

  7. [Application of aspartic acid as a non-specific binding inhibitor in the enrichment of phosphopeptides with titanium dioxide].

    Science.gov (United States)

    Chi, Ming; Bi, Wei; Lu, Zhuang; Song, Lina; Jia, Wei; Zhang, Yangjun; Qian, Xiaohong; Cai, Yun

    2010-02-01

    Titanium dioxide (TiO2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays. However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues. These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity. Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS). In this study, aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide. Firstly, the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide. The effects with the presence of aspartic acid, glutamic acid and no-inhibitor in the enrichment systems were compared separately. The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment. Then, aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples. Besides, no contamination in the ion source occurred during the mass spectrometric analysis.

  8. Modeling N-methyl-D-aspartate-induced bursting in dopamine neurons.

    Science.gov (United States)

    Li, Y X; Bertram, R; Rinzel, J

    1996-03-01

    Burst firing of dopaminergic neurons of the substantia nigra pars compacta can be induced in vitro by the glutamate agonist N-methyl-D-aspartate. It has been suggested that the interburst hyperpolarization is due to Na+ extrusion by a ouabain-sensitive pump [Johnson et al. (1992) Science 258, 665-667]. We formulate and explore a theoretical model, with a minimal number of currents, for this novel mechanism of burst generation. This minimal model is further developed into a more elaborate model based on observations of additional currents and hypotheses about their spatial distribution in dopaminergic neurons [Hounsgaard (1992) Neuroscience 50, 513-518; Llinás et al. (1984) Brain Res. 294, 127-132]. Using the minimal model, we confirm that interaction between the regenerative, inward N-methyl-D-aspartate-mediated current and the outward Na(+)-pump current is sufficient to generate the slow oscillation (approximately 0.5 Hz) underlying the burst. The negative-slope region of the N-methyl-D-aspartate channel's current-voltage relation is indispensable for this slow rhythm generation. The time-scale of Na(+)-handling determines the burst's slow frequency. Moreover, we show that, given the constraints of sodium handling, such bursting is best explained mechanistically by using at least two spatial, cable-like compartments: a soma where action potentials are produced and a dendritic compartment where the slow rhythm is generated. Our result is consistent with recent experimental evidence that burst generation originates in distal dendrites [Seutin et al. (1994) Neuroscience 58, 201-206]. Responses of the model to a number of electrophysiological and pharmacological stimuli are consistent with known responses observed under similar conditions. These include the persistence of the slow rhythm when the tetrodotoxin-sensitive Na+ channel is blocked and when the soma is voltage-clamped at -60 mV. Using our more elaborate model, we account for details of the observed

  9. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    Science.gov (United States)

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  10. Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

    Science.gov (United States)

    Headey, Stephen J; Macaskill, Ursula K; Wright, Michele A; Claridge, Jolyon K; Edwards, Patrick J B; Farley, Peter C; Christeller, John T; Laing, William A; Pascal, Steven M

    2010-08-27

    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

  11. Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

    OpenAIRE

    Boehm, M F; Bada, J L

    1984-01-01

    The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated tem...

  12. Hypoglycemia in type 1 diabetic pregnancy: role of preconception insulin aspart treatment in a randomized study

    DEFF Research Database (Denmark)

    Heller, Simon; Damm, Peter; Mersebach, Henriette;

    2010-01-01

    OBJECTIVE A recent randomized trial compared prandial insulin aspart (IAsp) with human insulin in type 1 diabetic pregnancy. The aim of this exploratory analysis was to investigate the incidence of severe hypoglycemia during pregnancy and compare women enrolled preconception with women enrolled...... during early pregnancy. RESEARCH DESIGN AND METHODS IAsp administered immediately before each meal was compared with human insulin administered 30 min before each meal in 99 subjects (44 to IAsp and 55 to human insulin) randomly assigned preconception and in 223 subjects (113 for IAsp and 110 for human...... insulin) randomly assigned in early pregnancy (...

  13. Prevalence of serum N-methyl-D-aspartate receptor autoantibodies in refractory psychosis

    OpenAIRE

    Beck*, Katherine Emma; Lally, John Alexander; Shergill, Sukhwinder S.; Bloomfield, Michael A.P.; MacCabe, James Hunter; Gaughran, Fiona Patricia; Howes, Oliver

    2015-01-01

    N-methyl-d-aspartate receptor (NMDA-R) autoantibodies have been reported in people with acute psychosis. We hypothesised that their presence may be implicated in the aetiology of treatment-refractory psychosis. We sought to ascertain the point prevalence of NMDA-R antibody positivity in patients referred to services for treatment-refractory psychosis. We found that 3 (7.0%) of 43 individuals had low positive NMDA-R antibody titres. This suggests that NMDA-R autoantibodies are unlikely to acco...

  14. Inclusion Behavior of Dimer b-Cyclodextrin Bridged with Aspartic Acid Derivative

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The b-cyclodextrin (CD) dimer bridged with aspartic acid (ASP) derivative, FITC-ASP(NH-b-CD)2 (Host, FITC=fluorescein-4-isothiocyanate), was synthesized. Fluorescence polarization study showed that the novel host formed an inclusion compound, [FITC-ASP(NH-b-CD)2]ATA, for which Kd was determined to be 5.0×10-6 mol/L by Beacon 2000 Analyzer, when ATA (Guest) = Adm-Trp-Arg-Arg-NH2 (Adm = 1-adamantanecarboxylic acid, Trp = tryptophan, Arg = arginine), where Kd is the dissociation constant in aqueous solution at 298 K.

  15. Existence of an Endogenous Glutamate and Aspartate Transporter in Chinese Hamster Ovary Cells

    Institute of Scientific and Technical Information of China (English)

    Xunhe JI; Yuhua JIN; Yaoyue CHEN; Chongyong LI; Lihe GUO

    2007-01-01

    Chinese hamster ovary cells show endogenous high-affinity Na+-dependent glutamate transport activity. This transport activity is kinetically similar to a glutamate transporter family strategically expressed in the central nervous system and is pharmacologically unlike glutamate transporter-1 or excitatory amino acid carrier 1. The cDNA of a glutamate/aspartate transporter (GLAST)-like transporter was obtained and analyzed. The deduced amino acid sequence showed high similarity to human, mouse, and rat GLAST. We concluded that a GLAST-like glutamate transporter exists in Chinese hamster ovary cells that might confer the endogenous high-affinity Na+-dependent glutamate transport activity evident in these cells.

  16. Aspartic acid racemization in dentin of the third molar for age estimation of the Chaoshan population in South China.

    Science.gov (United States)

    Chen, Shisheng; Lv, Yanyi; Wang, Dian; Yu, Xiaojun

    2016-09-01

    Aspartic acid racemization in teeth has been increasingly used to estimate chronological age with a considerably high accuracy in forensic practice. The Chaoshan population in South China is relatively isolated in geography, and has specific lifestyle and dietary inhibits. It is still unknown whether this method is suitable for this population. The aim of this study was to analyze the relationship between chronological age and the d/l aspartic acid ratio in dentin in the third molar tooth of the Chaoshan population. Fifty-eight non-carious third molar teeth (31 mandibles and 27 maxillae), from 58 living individuals of known age (24 males and 34 females), were retrieved. Dentin was extracted from these teeth. The d- and l-aspartic acids in dentins were separated and detected by high performance liquid chromatography (HPLC). Linear regression was performed between the d/l aspartic acid ratio of dentins and chronological age. Results showed that the correlation coefficient (r) was 0.969, and the mean absolute error (MAE) was 2.19 years, its standard deviation (SD) was ±1.53 years, indicating excellent correlation. There was no significant difference in racemization rates of dentin between sexes (P=0.113, F=2.6), or between mandibles and maxillae (P=0.964, F=0.000). Results indicate that the ratio of the d and l forms of aspartic acid of dentins, in the third molar, is closely correlated with chronological age, special lifestyle do no obviously affect the accuracy of the age estimations by aspartic acid racemization of the dentin in the third molar and that aspartic acid racemization in the third molar dentin can be used as an accurate method to estimate chronological age in the Chaoshan population in South China.

  17. Use of short-acting insulin aspart in managing older people with diabetes.

    Science.gov (United States)

    Marouf, Eltayeb; Sinclair, Alan J

    2009-01-01

    Type 2 diabetes mellitus affects 5.9% of the world adult population, with older people and some ethnic groups disproportionately affected. Treatment of older people with diabetes differs in many ways from that in younger adults since the majority have type 2 disease and are at particular risk of macrovascular rather than disabling microvascular disease. Insulin therapy, the most effective of diabetes medications, can reduce any level of elevated HBA1c if used in adequate doses. However, some clinicians are often reluctant to initiate insulin therapy in older people with diabetes mainly out of their concerns about adverse reactions to insulin, particularly hypoglycemia. There is evidence suggesting that insulin aspart appears to act similarly to regular human insulin in older people with type 2 diabetes mellitus. Insulin aspart can be used in the treatment of older people with diabetes, but this should be individualized. There is evidence that it improves postprandial glucose control, improves long-term metabolic control, reduces risk of major nocturnal hypoglycemia and increases patient satisfaction compared with soluble insulin.

  18. 7-hydroxycalamenene Effects on Secreted Aspartic Proteases Activity and Biofilm Formation of Candida spp.

    Science.gov (United States)

    Azevedo, Mariana M. B.; Almeida, Catia A.; Chaves, Francisco C. M.; Rodrigues, Igor A.; Bizzo, Humberto R.; Alviano, Celuta S.; Alviano, Daniela S.

    2016-01-01

    Background: The 7-hydroxycalamenenene-rich essential oil (EO) obtained from the leaves of Croton cajucara (red morphotype) have been described as active against bacteria, protozoa, and fungi species. In this work, we aimed to evaluate the effectiveness of 7-hydroxycalamenenene against Candida albicans and nonalbicans species. Materials and Methods: C. cajucara EO was obtained by hydrodistillation and its major compound, 7-hydroxycalamenene, was purified using preparative column chromatography. The anti-candidal activity was investigated by minimum inhibitory concentration (MIC) and secreted aspartic proteases (SAP) and biofilm inhibition assays. Results: 7-hydroxycalamenene (98% purity) displayed anti-candidal activity against all Candida species tested. Higher activity was observed against Candida dubliniensis, Candida parapsilosis and Candida albicans, showing MIC values ranging from 39.06 μg/ml to 78.12 μg/ml. The purified 7-hydroxycalamenene was able to inhibit 58% of C. albicans ATCC 36801 SAP activity at MIC concentration (pH 7.0). However, 7-hydroxycalamenene demonstrated poor inhibitory activity on C. albicans ATCC 10231 biofilm formation even at the highest concentration tested (2500 μg/ml). Conclusion: The bioactive potential of 7-hydroxycalamenene against planktonic Candida spp. further supports its use for the development of antimicrobials with anti-candidal activity. SUMMARY Croton cajucara Benth. essential oil provides high amounts of 7-hydroxycalamenene7-Hydroxycalameneneisolated from C. cajucarais active against Candida spp7-Hydroxycalameneneinhibits C. albicans aspartic protease activity7-Hydroxycalamenene was not active against C. albicans biofilm formation. Figure PMID:27019560

  19. Nanostructured aluminium oxide powders obtained by aspartic acid-nitrate gel-combustion routes

    Energy Technology Data Exchange (ETDEWEB)

    Gardey Merino, Maria Celeste, E-mail: mcgardey@frm.utn.edu.a [Laboratorio de Investigaciones y Servicios Ambientales Mendoza (LISAMEN) - CCT - CONICET, Avda. Ruiz Leal s/n, Parque Gral. San Martin, (M5502IRA) Ciudad de Mendoza, Prov. de Mendoza (Argentina); Grupo CLIOPE, Universidad Tecnologica Nacional - Facultad Regional Mendoza, Rodriguez 273, (M5502AJE) Ciudad de Mendoza, Prov. de Mendoza (Argentina); Lascalea, Gustavo E. [Laboratorio de Investigaciones y Servicios Ambientales Mendoza (LISAMEN) - CCT - CONICET, Avda. Ruiz Leal s/n, Parque Gral. San Martin, (M5502IRA) Ciudad de Mendoza, Prov. de Mendoza (Argentina); Sanchez, Laura M. [CINSO (Centro de Investigaciones en Solidos), CITEFA - CONICET, J.B. de La Salle 4397, (B1603ALO) Villa Martelli, Prov. de Buenos Aires (Argentina); Vazquez, Patricia G. [Centro de Investigacion y Desarrollo en Ciencias Aplicadas ' Dr. Jorge J. Ronco' (CINDECA), CONICET, Universidad Nacional de La Plata, Calle 47 nro. 257, (B1900AJK) La Plata, Prov. de Buenos Aires (Argentina); Cabanillas, Edgardo D. [CONICET and Centro Atomico Constituyentes, Comision Nacional de Energia Atomica, Gral. Paz 1499, (1650) San Martin, Prov. de Buenos Aires (Argentina); Lamas, Diego G. [CINSO (Centro de Investigaciones en Solidos), CITEFA - CONICET, J.B. de La Salle 4397, (B1603ALO) Villa Martelli, Prov. de Buenos Aires (Argentina)

    2010-04-16

    In this work, two new gel-combustion routes for the synthesis of Al{sub 2}O{sub 3} nanopowders with aspartic acid as fuel are presented. The first route is a conventional stoichiometric process, while the second one is a non-stoichiometric, pH-controlled process. These routes were compared with similar synthesis procedures using glycine as fuel, which are well-known in the literature. The samples were calcined in air at different temperatures, in a range of 600-1200 {sup o}C. They were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy and BET specific surface area. Different phases were obtained depending on the calcination temperature: amorphous, {gamma} (metastable) or {alpha} (stable). The amorphous-to-{gamma} transition was found for calcination temperatures in the range of 700-900 {sup o}C, while the {gamma}-to-{alpha} one was observed for calcination temperatures of 1100-1200 {sup o}C. The retention of the metastable {gamma} phase is probably due to a crystallite size effect. It transforms to the {alpha} phase after the crystallite size increases over a critical size during the calcination process at 1200 {sup o}C. The highest BET specific surface areas were obtained for both nitrate-aspartic acid routes proposed in this work, reaching values of about 50 m{sup 2}/g.

  20. Studies of the radioprotective properties of nicotinyl compounds, aspartic acid, glutamic acid and methionine

    International Nuclear Information System (INIS)

    Radioprotective properties of sodium salts of nicotinyl aspartic acid, nicotinyl methionyl aspartic acid and nicotinyl glutamic acid were tested in mice (NMRI). Experimental animals were irradiated by rayage (9,5 Gy). Parameters were: survival rate, peritoneal fluid cell count, weight and DNA concentration of spleen, hepatic DNA polymerase activity and rate of protein synthesis, lactate dehydrogenase activity in serum, maltase, sucrase and leucine aminopeptidase activitiy in duodenum and jejunum. Following results were obtained: 1. There was no significant difference in survival rate of treated and untreated animals. In treated animals only a short prolongation of survival time was observed. 2. After irradiation a quick reduction of splenic weight and DNA concentration was measured. 3. A reduction of DNA polymerase activity in liver was observed in treated and untreated mice. The rate of hepatic protein synthesis was similar in all animals. A final decrease was observed. 4. Variable activities of maltase, sucrase and leucine aminopeptidase activity in duodenum and jejunum indicated no radioprotective effect of tested substances. In conclusion of these results the tested substances show no significant radioprotective properties. (orig.)

  1. Membrane topology of aspartate:alanine antiporter AspT from Comamonas testosteroni.

    Science.gov (United States)

    Fujiki, Takashi; Nanatani, Kei; Nishitani, Kei; Yagi, Kyoko; Ohnishi, Fumito; Yoneyama, Hiroshi; Uchida, Takafumi; Nakajima, Tasuku; Abea, Keietsu

    2007-01-01

    We cloned the aspT gene encoding the L-aspartate:L-alanine antiporter AspTCt in Comamonas testosteroni genomic DNA. Analysis of the nucleotide sequence revealed that C. testosteroni has an asp operon containing aspT upstream of the l-aspartate 4-decarboxylase gene, and that the gene order of the asp operon of C. testosteroni is the inverse of that of Tetragenococcus halophilus. We used proteoliposomes to confirm the transport processes of AspTCt. To elucidate the two-dimensional structure of AspTCt, we analysed its membrane topology by means of alkaline phosphatase (PhoA) and beta-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing approximately 200 amino acid residues between TM4 and TM5, a cytoplasmic N-terminus, and a periplasmic C-terminus. These results suggest that the orientation of the N-terminus of AspTCt differs from that of tetragenococcal AspT, even though these two AspT orthologues catalyse the same transport reactions. PMID:17158863

  2. [Aspartic Acid Generated in the Process of Chlorination Disinfection By-product Dichloroacetonitrile].

    Science.gov (United States)

    Ding, Chun-sheng; Li, Nai-jun; Zhang, Tao; Zhang, Meng-qing

    2016-05-15

    In this study, a method was developed for the determination of dichloroacetonitrile (DCAN) in drinking water by liquid- liquid micro-extraction and gas chromatography/mass spectrometry ( LLE-GC/MS), which used 1,2-dibromopropane as the internal standard and methyl tertiary butyl ether (MTBE) as the extractant for high accuracy. The aspartic acid was used as the precursor of the DCAN formation during chlorination and the influencing factors were evaluated. The formation mechanism of DCAN was also discussed. The results showed that the DCAN amount increased with the increase of pH value under the neutral and acidic conditions, however, the amount of DCAN decreased with the increase of pH value under the alkali condition. And the final amount of DCAN under the alkali condition was much less than that under the neutral and acidic conditions. It was also found that the DCAN amount increased with the increase of chlorine addition, while the temperature in the range of 10-30°C had little influence on the DCAN formation. The formation process of the DCAN from aspartic acid by chlorination included seven steps, such as substitution, decarboxylation, oxidation, etc and ultimately formed DCAN. PMID:27506037

  3. [Aspartic Acid Generated in the Process of Chlorination Disinfection By-product Dichloroacetonitrile].

    Science.gov (United States)

    Ding, Chun-sheng; Li, Nai-jun; Zhang, Tao; Zhang, Meng-qing

    2016-05-15

    In this study, a method was developed for the determination of dichloroacetonitrile (DCAN) in drinking water by liquid- liquid micro-extraction and gas chromatography/mass spectrometry ( LLE-GC/MS), which used 1,2-dibromopropane as the internal standard and methyl tertiary butyl ether (MTBE) as the extractant for high accuracy. The aspartic acid was used as the precursor of the DCAN formation during chlorination and the influencing factors were evaluated. The formation mechanism of DCAN was also discussed. The results showed that the DCAN amount increased with the increase of pH value under the neutral and acidic conditions, however, the amount of DCAN decreased with the increase of pH value under the alkali condition. And the final amount of DCAN under the alkali condition was much less than that under the neutral and acidic conditions. It was also found that the DCAN amount increased with the increase of chlorine addition, while the temperature in the range of 10-30°C had little influence on the DCAN formation. The formation process of the DCAN from aspartic acid by chlorination included seven steps, such as substitution, decarboxylation, oxidation, etc and ultimately formed DCAN.

  4. Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate β-Decarboxylase Activity and Application to l-Alanine Production

    OpenAIRE

    Fusee, Murray C.; Weber, Jennifer E.

    1984-01-01

    Whole cells of Pseudomonas dacunhae containing l-aspartate β-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate β-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as...

  5. The effect of preirradiation application of aspartic acid salts on hemopoietic recovery in X-irradiated mice

    International Nuclear Information System (INIS)

    The possibility of radioprotective action of K and Mg aspartate administered in tap water for ten days prior to X-irradiation was investigated in male mice of the strain C 57 Bl/10. In normal animals, thymus weight was found to be increased by 10-day treatment with K and Mg aspartate. The postirradiation regeneration of spleen weight and incorporation of radioactive iron into the spleen and femoral marrow following sublethal irradiation was favorably modified by the treatment used. Pretreatment of mice with K and Mg aspartate delays the onset of early deaths at irradiation with an absolutely lethal X-ray exposure and raises the percentage of surviving animals after nearly lethal exposures. (orig.)

  6. Synthesis of a stable gold hydrosol by the reduction of chloroaurate ions by the amino acid, aspartic acid

    Indian Academy of Sciences (India)

    Saikat Mandal; P R Selvakannan; Sumant Phadtare; Renu Pasricha; Murali Sastry

    2002-10-01

    Development of reliable protocols for the synthesis of nanoparticles of well-defined sizes and good monodispersity is an important aspect of nanotechnology. In this paper, we present details of the synthesis of gold nanoparticles of good monodispersity by the reduction of aqueous chloroaurate ions by the amino acid, aspartic acid. The colloidal gold solution thus formed is extremely stable in time, indicating electrostatic stabilization via nanoparticle surface-bound amino acid molecules. This observation has been used to modulate the size of the gold nanoparticles in solution by varying the molar ratio of chloroaurate ions to aspartic acid in the reaction medium. Characterization of the aspartic acid-reduced gold nanoparticles was carried out by UV-visible spectroscopy, thermogravimetric analysis and transmission electron microscopy. The use of amino acids in the synthesis and stabilization of gold nanoparticle in water has important implications in the development of new protocols for generation of bioconjugate materials.

  7. RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

    Directory of Open Access Journals (Sweden)

    Rui Cruz

    2014-08-01

    Full Text Available Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a

  8. Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

    OpenAIRE

    Kazuma Ogawa; Atsushi Ishizaki; Kenichiro Takai; Yoji Kitamura; Tatsuto Kiwada; Kazuhiro Shiba; Akira Odani

    2013-01-01

    (68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA...

  9. Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine]:A Non-viral Polymer with Potential for DNA Delivery

    Institute of Scientific and Technical Information of China (English)

    Zhi YANG; Gu Ping TANG

    2004-01-01

    A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).

  10. Immobilization of Escherichia coli Cells Containing Aspartase Activity with Polyurethane and Its Application for l-Aspartic Acid Production

    OpenAIRE

    Fusee, Murray C.; Swann, Wayne E.; Calton, Gary J.

    1981-01-01

    Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37°C as compared to 25°C operating temperature. The p...

  11. Synthesis, Characterization, and Antimicrobial Activities of Coordination Compounds of Aspartic Acid

    Directory of Open Access Journals (Sweden)

    T. O. Aiyelabola

    2016-01-01

    Full Text Available Coordination compounds of aspartic acid were synthesized in basic and acidic media, with metal ligand M : L stoichiometric ratio 1 : 2. The complexes were characterized using infrared, electronic and magnetic susceptibility measurements, and mass spectrometry. Antimicrobial activity of the compounds was determined against three Gram-positive and three Gram-negative bacteria and one fungus. The results obtained indicated that the availability of donor atoms used for coordination was a function of the pH of the solution in which the reaction was carried out. This resulted in varying geometrical structures for the complexes. The compounds exhibited a broad spectrum of activity and in some cases better activity than the standard.

  12. Opioid analgesics as noncompetitive N-methyl-D-aspartate (NMDA) antagonists

    DEFF Research Database (Denmark)

    Ebert, B; Thorkildsen, C; Andersen, S;

    1998-01-01

    Much evidence points to the involvement of N-methyl-D-aspartate (NMDA) receptors in the development and maintainance of neuropathic pain. In neuropathic pain, there is generally involved a presumed opioid-insensitive component, which apparently can be blocked by NMDA receptor antagonists. However......, in order to obtain complete analgesia, a combination of an NMDA receptor antagonist and an opioid receptor agonist is needed. Recent in vitro data have demonstrated that methadone, ketobemidone, and dextropropoxyphene, in addition to being opioid receptor agonists, also are weak noncompetitive NMDA...... receptor antagonists. Clinical anecdotes suggest that the NMDA receptor antagonism of these opioids may play a significant role in the pharmacological action of these compounds; however, no clinical studies have been conducted to support this issue. In the present commentary, we discuss evidence...

  13. Expansion of the aspartate [beta]-semialdehyde dehydrogenase family: the first structure of a fungal ortholog

    Energy Technology Data Exchange (ETDEWEB)

    Arachea, B.T.; Liu, X.; Pavlovsky, A.G.; Viola, R.E. (Toledo)

    2010-08-13

    The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development.

  14. An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis.

    Science.gov (United States)

    Ge, Xiaochun; Dietrich, Charles; Matsuno, Michiyo; Li, Guojing; Berg, Howard; Xia, Yiji

    2005-03-01

    The components and pathways that regulate and execute developmental cell death programmes in plants remain largely unknown. We have found that the PROMOTION OF CELL SURVIVAL 1 (PCS1) gene in Arabidopsis, which encodes an aspartic protease, has an important role in determining the fate of cells in embryonic development and in reproduction processes. The loss-of-function mutation of PCS1 causes degeneration of both male and female gametophytes and excessive cell death of developing embryos. Conversely, ectopic expression of PCS1 causes the septum and stomium cells that normally die in the anther wall to survive instead, leading to a failure in anther dehiscence and male sterility. PCS1 provides a new avenue for understanding the mechanisms of the programmed cell death processes that are associated with developmental pathways in plants and makes available a useful tool for engineering the male sterility trait for hybrid seed production.

  15. Clinical use of the co-formulation of insulin degludec and insulin aspart

    DEFF Research Database (Denmark)

    Kumar, A; Awata, T; Bain, S C;

    2016-01-01

    AIMS: To provide a review of the available data and practical use of insulin degludec with insulin aspart (IDegAsp). Premixed insulins provide basal and prandial glucose control; however, they have an intermediate-acting prandial insulin component and do not provide as effective basal coverage...... as true long-acting insulins, owing to the physicochemical incompatibility of their individual components, coupled with the inflexibility of adjustment. The molecular structure of the co-formulation of IDegAsp, a novel insulin preparation, allows these two molecules to coexist without affecting...... (HbA1c ) to current modern insulins, but with lower risk of nocturnal hypoglycaemia. In prior insulin users, glycaemic control was achieved with lower or equal insulin doses vs. other basal+meal-time or premix insulin regimens. In insulin-naïve patients with T2DM, IDegAsp can be started once or twice...

  16. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne;

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  17. Application and appreciation of chemical sand fixing agent-poly (aspartic acid) and its composites

    Energy Technology Data Exchange (ETDEWEB)

    Yang Jun; Cao Hui; Wang Fang [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China); Tan Tianwei [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China)], E-mail: twtan@mail.buct.edu.cn

    2007-12-15

    The sand fixing agent-poly (aspartic acid) (PASP) and its composites were applied in the field by two forms (spraying around by PASP solution and PASP powder directly). It was found that the sand fixing effect in powder form was not as good as in solution form, but it was more practical in dry region. It needed 9, 6 and 7 days for PASP, xanthan gum-PASP (X2) and ethyl cellulose-PASP (E3) to attain the maximal mechanical strength after they were applied, respectively. The sand fixing effect decreased when the material was subjected to repeated hydration-dehydration cycles and the material had no negative influence on plant growth. The PASP and its composites had water-retaining ability and could reduce the water evaporation. - The sand fixing agent was applied in powder form and it had no negative influence on plant growth.

  18. Application and appreciation of chemical sand fixing agent-poly (aspartic acid) and its composites

    International Nuclear Information System (INIS)

    The sand fixing agent-poly (aspartic acid) (PASP) and its composites were applied in the field by two forms (spraying around by PASP solution and PASP powder directly). It was found that the sand fixing effect in powder form was not as good as in solution form, but it was more practical in dry region. It needed 9, 6 and 7 days for PASP, xanthan gum-PASP (X2) and ethyl cellulose-PASP (E3) to attain the maximal mechanical strength after they were applied, respectively. The sand fixing effect decreased when the material was subjected to repeated hydration-dehydration cycles and the material had no negative influence on plant growth. The PASP and its composites had water-retaining ability and could reduce the water evaporation. - The sand fixing agent was applied in powder form and it had no negative influence on plant growth

  19. D-Aspartate drinking solution alleviates pain and cognitive impairment in neuropathic mice.

    Science.gov (United States)

    Palazzo, Enza; Luongo, Livio; Guida, Francesca; Marabese, Ida; Romano, Rosaria; Iannotta, Monica; Rossi, Francesca; D'Aniello, Antimo; Stella, Luigi; Marmo, Federica; Usiello, Alessandro; de Bartolomeis, Andrea; Maione, Sabatino; de Novellis, Vito

    2016-07-01

    D-Aspartate (D-Asp) is a free D-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-D-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether D-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with D-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. D-Asp also restored the levels of extracellular D-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike D-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that D-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice. PMID:27115160

  20. Anti-N-methyl-D-aspartate receptor encephalitis presenting with acute psychosis in a preteenage girl: a case report

    Directory of Open Access Journals (Sweden)

    Maggina Paraskevi

    2012-07-01

    Full Text Available Abstract Introduction Anti-N-methyl-D-aspartate receptor (anti-NMDAR encephalitis is a rare, newly defined autoimmune clinical entity that presents with atypical clinical manifestations. Most patients with anti-N-methyl-D-aspartate receptor encephalitis develop a progressive illness from psychosis into a state of unresponsiveness, with catatonic features often associated with abnormal movements and autonomic instability. This is the first report of anti-N-methyl-D-aspartate receptor encephalitis in a Greek pediatric hospital. Case presentation An 11-year-old Greek girl presented with clinical manifestations of acute psychosis. The differential diagnosis included viral encephalitis. The presence of a tumor usually an ovarian teratoma, a common clinical finding in many patients, was excluded. Early diagnosis and prompt immunotherapy resulted in full recovery up to one year after the initial diagnosis. Conclusion Acute psychosis is a rare psychiatric presentation in children, diagnosed only after possible organic syndromes that mimic acute psychosis are excluded, including anti-N-methyl-D-aspartate receptor receptor encephalitis. Pediatricians, neurologists and psychiatrists should consider this rare clinical syndrome, in order to make an early diagnosis and instigate appropriate treatment to maximize neurological recovery.

  1. The Asc locus for resistance to Alternaria stem canker in tomato does not encode the enzyme aspartate carbamoyltransferase

    NARCIS (Netherlands)

    Overduin, Bert; Hogenhout, Saskia A.; Biezen, Erik A. van der; Haring, Michel A.; Nijkamp, H. John J.; Hille, Jacques

    1993-01-01

    The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (ACTase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The P

  2. Aspartic acid racemization rate in narwhal (Monodon monoceros) eye lens nuclei estimated by counting of growth layers in tusks

    DEFF Research Database (Denmark)

    Garde, Eva; Heide-Jørgensen, Mads Peter; Ditlevsen, Susanne;

    2012-01-01

    ) technique has been used in age estimation studies of cetaceans, including narwhals. The purpose of this study was to estimate a species-specific racemization rate for narwhals by regressing aspartic acid D/L ratios in eye lens nuclei against growth layer groups in tusks (n=9). Two racemization rates were...

  3. Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations.

    Science.gov (United States)

    Lenton, Samuel; Walsh, Danielle L; Rhys, Natasha H; Soper, Alan K; Dougan, Lorna

    2016-07-21

    Halophilic organisms have adapted to survive in high salt environments, where mesophilic organisms would perish. One of the biggest challenges faced by halophilic proteins is the ability to maintain both the structure and function at molar concentrations of salt. A distinct adaptation of halophilic proteins, compared to mesophilic homologues, is the abundance of aspartic acid on the protein surface. Mutagenesis and crystallographic studies of halophilic proteins suggest an important role for solvent interactions with the surface aspartic acid residues. This interaction, between the regions of the acidic protein surface and the solvent, is thought to maintain a hydration layer around the protein at molar salt concentrations thereby allowing halophilic proteins to retain their functional state. Here we present neutron diffraction data of the monomeric zwitterionic form of aspartic acid solutions at physiological pH in 0.25 M and 2.5 M concentration of potassium chloride, to mimic mesophilic and halophilic-like environmental conditions. We have used isotopic substitution in combination with empirical potential structure refinement to extract atomic-scale information from the data. Our study provides structural insights that support the hypothesis that carboxyl groups on acidic residues bind water more tightly under high salt conditions, in support of the residue-ion interaction model of halophilic protein stabilisation. Furthermore our data show that in the presence of high salt the self-association between the zwitterionic form of aspartic acid molecules is reduced, suggesting a possible mechanism through which protein aggregation is prevented. PMID:27327567

  4. Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid

    NARCIS (Netherlands)

    Sivan, S.-S.; Wachtel, E.; Tsitron, E.; Sakkee, N.; Ham, F. van der; Groot, J.de; Roberts, S.; Maroudas, A.

    2008-01-01

    Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We

  5. Stimulation of the N-methyl-D-aspartate receptor has a trophic effect on differentiating cerebellar granule cells

    DEFF Research Database (Denmark)

    Balázs, R; Hack, N; Jørgensen, Ole Steen

    1988-01-01

    N-methyl-D-aspartate (NMDA) supplementation of cerebellar cultures enriched in granule neurones (about 90%) prevented the extensive cell loss which occurs when cultivation takes place, in serum containing media, in the presence of 'low' K+ (5-15 mM). Estimation of tetanus toxin receptors and N...

  6. Quantification of the novel N-methyl-d-aspartate receptor ligand [11C]GMOM in man.

    Science.gov (United States)

    van der Doef, Thalia F; Golla, Sandeep Sv; Klein, Pieter J; Oropeza-Seguias, Gisela M; Schuit, Robert C; Metaxas, Athanasios; Jobse, Ellen; Schwarte, Lothar A; Windhorst, Albert D; Lammertsma, Adriaan A; van Berckel, Bart Nm; Boellaard, Ronald

    2016-06-01

    [(11)C]GMOM (carbon-11 labeled N-(2-chloro-5-thiomethylphenyl)-N'-(3-[(11)C]methoxy-phenyl)-N'-methylguanidine) is a PET ligand that binds to the N-methyl-d-aspartate receptor with high specificity and affinity. The purpose of this first in human study was to evaluate kinetics of [(11)C]GMOM in the healthy human brain and to identify the optimal pharmacokinetic model for quantifying these kinetics, both before and after a pharmacological dose of S-ketamine. Dynamic 90 min [(11)C]GMOM PET scans were obtained from 10 subjects. In six of the 10 subjects, a second PET scan was performed following an S-ketamine challenge. Metabolite corrected plasma input functions were obtained for all scans. Regional time activity curves were fitted to various single- and two-tissue compartment models. Best fits were obtained using a two-tissue irreversible model with blood volume parameter. The highest net influx rate (Ki) of [(11)C]GMOM was observed in regions with high N-methyl-d-aspartate receptor density, such as hippocampus and thalamus. A significant reduction in the Ki was observed for the entire brain after administration of ketamine, suggesting specific binding to the N-methyl-d-aspartate receptors. This initial study suggests that the [(11)C]GMOM could be used for quantification of N-methyl-d-aspartate receptors. PMID:26661185

  7. Cortical N-acetyl aspartate is a predictor of long-term clinical disability in multiple sclerosis

    DEFF Research Database (Denmark)

    Wu, Xingchen; Hanson, Lars G.; Skimminge, Arnold Jesper Møller;

    2014-01-01

    Objective: To evaluate the prognostic value of the cortical N-acetyl aspartate to creatine ratio (NAA/Cr) in early relapsing-remitting multiple sclerosis (RRMS). Methods: Sixteen patients with newly diagnosed RRMS were studied by serial MRI and MR spectroscopic imaging (MRSI) once every 6 months ...

  8. Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Kwangseon Jung

    Full Text Available Ultraviolet A (UVA irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs. Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA.

  9. Kinetic simulation of malate-aspartate and citrate-pyruvate shuttles in association with Krebs cycle.

    Science.gov (United States)

    Korla, Kalyani; Vadlakonda, Lakshmipathi; Mitra, Chanchal K

    2015-01-01

    In the present work, we have kinetically simulated two mitochondrial shuttles, malate-aspartate shuttle (used for transferring reducing equivalents) and citrate-pyruvate shuttle (used for transferring carbon skeletons). However, the functions of these shuttles are not limited to the points mentioned above, and they can be used in different arrangements to meet different cellular requirements. Both the shuttles are intricately associated with Krebs cycle through the metabolites involved. The study of this system of shuttles and Krebs cycle explores the response of the system in different metabolic environments. Here, we have simulated these subsets individually and then combined them to study the interactions among them and to bring out the dynamics of these pathways in focus. Four antiports and a pyruvate pump were modelled along with the metabolic reactions on both sides of the inner mitochondrial membrane. Michaelis-Menten approach was extended for deriving rate equations of every component of the system. Kinetic simulation was carried out using ordinary differential equation solver in GNU Octave. It was observed that all the components attained steady state, sooner or later, depending on the system conditions. Progress curves and phase plots were plotted to understand the steady state behaviour of the metabolites involved. A comparative analysis between experimental and simulated data show fair agreement thus validating the usefulness and applicability of the model.

  10. Effects of Zinc Magnesium Aspartate (ZMA Supplementation on Training Adaptations and Markers of Anabolism and Catabolism

    Directory of Open Access Journals (Sweden)

    Almada Anthony

    2004-12-01

    Full Text Available Abstract This study examined whether supplementing the diet with a commercial supplement containing zinc magnesium aspartate (ZMA during training affects zinc and magnesium status, anabolic and catabolic hormone profiles, and/or training adaptations. Forty-two resistance trained males (27 ± 9 yrs; 178 ± 8 cm, 85 ± 15 kg, 18.6 ± 6% body fat were matched according to fat free mass and randomly assigned to ingest in a double blind manner either a dextrose placebo (P or ZMA 30–60 minutes prior to going to sleep during 8-weeks of standardized resistance-training. Subjects completed testing sessions at 0, 4, and 8 weeks that included body composition assessment as determined by dual energy X-ray absorptiometry, 1-RM and muscular endurance tests on the bench and leg press, a Wingate anaerobic power test, and blood analysis to assess anabolic/catabolic status as well as markers of health. Data were analyzed using repeated measures ANOVA. Results indicated that ZMA supplementation non-significantly increased serum zinc levels by 11 – 17% (p = 0.12. However, no significant differences were observed between groups in anabolic or catabolic hormone status, body composition, 1-RM bench press and leg press, upper or lower body muscular endurance, or cycling anaerobic capacity. Results indicate that ZMA supplementation during training does not appear to enhance training adaptations in resistance trained populations.

  11. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    Science.gov (United States)

    Torruella, M; Gordon, K; Hohn, T

    1989-10-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed. PMID:2684630

  12. Evaluation of poly (aspartic acid sodium salt) as a draw solute for forward osmosis.

    Science.gov (United States)

    Gwak, Gimun; Jung, Bokyung; Han, Sungsoo; Hong, Seungkwan

    2015-09-01

    Poly (aspartic acid sodium salt) (PAspNa) was evaluated for its potential as a novel draw solute in forward osmosis (FO). The inherent advantages of PAspNa, such as good water solubility, high osmotic pressure, and nontoxicity, were first examined through a series of physicochemical analyses and atomic-scale molecular dynamics simulations. Then, lab-scale FO tests were performed to evaluate its suitability in practical processes. Compared to other conventional inorganic solutes, PAspNa showed comparable water flux but significantly lower reverse solute flux, demonstrating its suitability as a draw solute. Moreover, fouling experiments using synthetic wastewater as a feed solution demonstrated that PAspNa reversely flowed to the feed side reduced inorganic scaling on the membrane active layer. The recyclability of PAspNa was studied using both nanofiltration (NF) and membrane distillation (MD) processes, and the results exhibited its ease of recovery. This research reported the feasibility and applicability of FO-NF or FO-MD processes using PAspNa for wastewater reclamation and brackish water desalination.

  13. Finding a Leucine in a Haystack: Searching the Proteome for ambigous Leucine-Aspartic Acid motifs

    KAUST Repository

    Arold, Stefan T.

    2016-01-25

    Leucine-aspartic acid (LD) motifs are short helical protein-protein interaction motifs involved in cell motility, survival and communication. LD motif interactions are also implicated in cancer metastasis and are targeted by several viruses. LD motifs are notoriously difficult to detect because sequence pattern searches lead to an excessively high number of false positives. Hence, despite 20 years of research, only six LD motif–containing proteins are known in humans, three of which are close homologues of the paxillin family. To enable the proteome-wide discovery of LD motifs, we developed LD Motif Finder (LDMF), a web tool based on machine learning that combines sequence information with structural predictions to detect LD motifs with high accuracy. LDMF predicted 13 new LD motifs in humans. Using biophysical assays, we experimentally confirmed in vitro interactions for four novel LD motif proteins. Thus, LDMF allows proteome-wide discovery of LD motifs, despite a highly ambiguous sequence pattern. Functional implications will be discussed.

  14. Predicting three-dimensional conformations of peptides constructed of only glycine, alanine, aspartic acid, and valine.

    Science.gov (United States)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  15. Formation of haloacetamides during chlorination of dissolved organic nitrogen aspartic acid

    Energy Technology Data Exchange (ETDEWEB)

    Chu Wenhai, E-mail: 1world1water@tongji.edu.cn [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai, 200092 (China); Gao Naiyun [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai, 200092 (China); Deng Yang, E-mail: yang.deng@upr.edu [Department of Civil Engineering and Surveying, University of Puerto Rico, P.O. Box 9041, Mayaguez, Puerto Rico, 00681-9041 (Puerto Rico)

    2010-01-15

    The stability of haloacetamides (HAcAms) such as dichloroacetamide (DCAcAm) and trichloroacetamide (TCAcAm) was studied under different experimental conditions. The yield of HAcAms during aspartic acid (Asp) chlorination was measured at different molar ratio of chlorine atom to nitrogen atom (Cl/N), pH and dissolved organic carbon (DOC) mainly consisted of humic acid (HA) mixture. Ascorbic acid showed a better capacity to prevent the decay of DCAcAm and TCAcAm than the other two dechlorinating agents, thiosulfate and sodium sulfite. Lower Cl/N favored the DCAcAm formation, implying that breakpoint chlorination might minimize its generation. The pH decrease could lower the concentration of DCAcAm but favored dichloroacetonitrile (DCAN) formation. DCAcAm yield was sensitive to the DOC due to higher chlorine consumption caused by HA mixture. Two possible pathways of DCAcAm formation during Asp chlorination were proposed. Asp was an important precursor of DCAN, DCAcAm and dichloroacetic acid (DCAA), and thus removal of Asp before disinfection may be a method to prevent the formation of DCAcAm, DCAN and DCAA.

  16. Effects of N-methyl-D-aspartate antagonists on different measures of motion sickness in cats.

    Science.gov (United States)

    Lucot, J B

    1998-11-15

    Because N-methyl-D-aspartate (NMDA) antagonists prevent cisplatin-induced emesis and NMDA receptors are in both emetic pathways and structures associated with the final common pathway for vomiting, they have the potential to be broad-spectrum antiemetics. This was evaluated by determining their effects on motion sickness in cats. The measures included the number vomiting, the number of symptom points, which reflect activity early in the final common path and the duration of the retch/vomit sequence, which reflects activity late in the path. Dextrorphan, ketamine and dextromethorphan decreased the number vomiting with the same rank order of potency as at NMDA receptors. Additional studies with 1,3-dio-tolylguaninidine (DTG) and haloperidol ruled out a role for sigma receptors. The NMDA antagonists produced a nonsignificant dose-dependent decrease in symptoms and had no effects on the duration of vomiting. They also produced motor abnormalities at the highest doses. The competitive antagonist LY 233053 also decreased the number vomiting without altering the duration. It produced a nonsignificant non-dose-dependent decrease in symptoms and had no effects on gross motor output. The results are consistent with a broad spectrum of antiemetic efficacy with at least a part of its action in the early to middle portions of the final common pathway for vomiting. Additional actions on the vestibular nuclei are possible. PMID:10052568

  17. Involvement of mitogen-activated protein kinase pathways in N-methyl-D-aspartate-induced excitotoxicity

    Institute of Scientific and Technical Information of China (English)

    Xiaorong Yang; Ping Sun; Huaping Qin; Rui Wang; Ye Wang; Ruihong Shi; Xin Zhao; Ce Zhang

    2011-01-01

    Previous studies have shown that mitogen-activated protein kinase (MAPK) signaling pathways are involved in N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. However, a systematic observation or analysis of the role of these various MAPK pathways in excitotoxicity processes does not exist. The present study further evaluated the role and contribution of three MAPK pathways extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK in an NMDA-mediated excitotoxicity model using MAPK-specific inhibitor. Results demonstrated that c-Jun N-terminal kinase inhibitor SP600125 and/or p38 MAPK inhibitor SB203580 inhibited NMDA-induced reduction in cell viability, as well as reduced NMDA-induced lactate dehydrogenase leakage and reactive oxygen species production. However, PD98059, an inhibitor of extracellular signal-regulated kinase, did not influence this model. Results demonstrated an involvement of c-Jun N-terminal kinase and p38 MAPK, but not extracellular signal-regulated kinase, in NMDA-mediated excitotoxicity in cortical neurons.

  18. Evaluation of poly (aspartic acid sodium salt) as a draw solute for forward osmosis.

    Science.gov (United States)

    Gwak, Gimun; Jung, Bokyung; Han, Sungsoo; Hong, Seungkwan

    2015-09-01

    Poly (aspartic acid sodium salt) (PAspNa) was evaluated for its potential as a novel draw solute in forward osmosis (FO). The inherent advantages of PAspNa, such as good water solubility, high osmotic pressure, and nontoxicity, were first examined through a series of physicochemical analyses and atomic-scale molecular dynamics simulations. Then, lab-scale FO tests were performed to evaluate its suitability in practical processes. Compared to other conventional inorganic solutes, PAspNa showed comparable water flux but significantly lower reverse solute flux, demonstrating its suitability as a draw solute. Moreover, fouling experiments using synthetic wastewater as a feed solution demonstrated that PAspNa reversely flowed to the feed side reduced inorganic scaling on the membrane active layer. The recyclability of PAspNa was studied using both nanofiltration (NF) and membrane distillation (MD) processes, and the results exhibited its ease of recovery. This research reported the feasibility and applicability of FO-NF or FO-MD processes using PAspNa for wastewater reclamation and brackish water desalination. PMID:26005789

  19. Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

    Directory of Open Access Journals (Sweden)

    MIRA D. MILISAVLJEVIĆ

    2009-06-01

    Full Text Available Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum aspartic protease (FeAPL1 in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation indicated the importance of the twelve cysteine residues for correct folding and functionality.

  20. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    Science.gov (United States)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  1. Properties of human liver cytosolic aspartate aminotransferase mRNAs generated by alternative polyadenylation site selection

    International Nuclear Information System (INIS)

    Human cytosolic aspartate aminotransferase (cAspAT) cDNA clones have been isolated from an adult human liver cDNA library. Among the clones, two cDNAs of 1,550 and 1,950 base pairs, respectively, have been characterized. These two cDNAs differ only in the lengths of their 3' noncoding regions and by the presence of one or two putative polyadenylation signals AATAAA. Northern blot analysis revealed two different mRNAs of 2.1 and 1.8 kbp in several human tissues, whereas Southern blot analysis suggested the existence of a single gene for the human cAspAT. The two mRNA species result from the alternative use of two polyadenylation signals. In the liver, the relative ratio of these mRNAs varies among different species and, in humans at least, during development. The properties of the two mRNAs were compared. The half-lives of the 2.1 and 1.8 kbp mRNAs, in the HepG2 cell line, are 8 and 12 h, respectively. The two mRNAs have similar and rather short poly(A) tracts of 20-50 nucleotides. Both mRNAs are capable of directing the in vitro synthesis of the cAspAT protein. The authors conclude that both the 2.1 and 1.8 kbp cAspAT mRNAs are functional and exhibit similar properties

  2. Effect of acute potassium-magnesium aspartate supplementation on ammonia concentrations during and after resistance training.

    Science.gov (United States)

    Tuttle, J L; Potteiger, J A; Evans, B W; Ozmun, J C

    1995-06-01

    This study examined the effects of aspartate supplementation (ASP) on plasma ammonia concentrations ([NH4+]) during and after a resistance training workout (RTW). Twelve male weight trainers were randomly administered ASP or vitamin C in a crossover, double blind protocol, each trial separated by 1 wk. ASP and vitamin C were given over a 2-hr period beginning 5 hr prior to the RTW. The RTW consisted of bench, incline, shoulder, and triceps presses, and biceps curls at 70% of one repetition maximum (1-RM). After the RTW a bench press test (BPT) to failure at 65% of 1-RM was used to assess performance. [NH4+] was determined preexercise, 20 and 40 min midworkout, immediately postexercise, and 15 min postexercise. Treatment-by-time ANOVAs, paired t tests, and contrast comparisons were used to identify mean differences. No significant differences were observed between treatments for [NH4+] or BPT. [NH4+] increased significantly from Pre to immediately postexercise for both the ASP and vitamin C trials. Acute ASP supplementation does not reduce [NH4+] during and after a high intensity RTW in weight trained subjects. PMID:7670449

  3. Disproportional exaggerated aspartate transaminase is a useful prognostic parameter in late leptospirosis

    Institute of Scientific and Technical Information of China (English)

    Ming-Ling Chang; Chih-Wei Yang; Jeng-Chang Chen; Yu-Pin Ho; Ming-Jeng Pan; Cheng-Hui Lin; Deng-Yn Lin

    2005-01-01

    AIM: To evaluate the hepatic dysfunction in leptospirosis is usually mild and resolved eventually. However,sequential follow-up of liver biochemical data remained lacking..METHODS: The biochemistry data and clinical symptoms of 11 sporadic patients were collected and analyzed, focusing on the impacts of leptospirosis upon liver biochemistry tests.RESULTS: The results disclosed that of the 11 cases, 5 or 45% died. The liver biochemistry data in the beginning of the disease course were only mildly elevated.Nevertheless, late exaggerated aspartate transaminase (AST)elevations were noted in three cases who finally died when compared with the typical course. Besides, significant higher AST/alanine transaminase (ALT) ratios (AARs) of the peak levels for transaminase were also noted in the cases who eventually succumbed. The mean±SD of AARs for the survival group and dead group were 5.65±2.27 (n = 5)and 1.86±0.64 (n = 6) respectively (P= 0.006). The ratios of the cases who finally died were all more than 3.0.Conversely, the survival group's ratios were less than 3.0.CONCLUSION: Serial follow-up of transaminase might provide evidence to predict some rare evolutions in leptospirosis. If AST elevated progressively without a concomitant change of ALT, it might indicate an acute disease course with ensuing death. Additionally, AAR is another prognostic parameter for leptospirosis. Once the value was higher than 3.0, a grave prognosis is inevitable.

  4. Synthesis, Micellization and Characterization of Novel Amphiphilic β-Cyclodextrin/Poly(L-aspartate) Copolymer

    Institute of Scientific and Technical Information of China (English)

    GUO Yan-ling; CUI Zhao-shan; HAN Min

    2013-01-01

    A novel amphiphilic β-cyclodextrin/poly(L-aspartate)(β-CD-PASP) copolymer was prepared by ringopening polymerization of polysuccinimide(PSI).This copolymer bears β-CD units along the macromolecular chain and the structure was characterized by infrared(IR) and proton nuclear magnetic resonance(1H NMR).The molecular weight of the copolymer was determined by gel permeation chromatography(GPC).The copolymer micelle were prepared by direct dissolution method.The critical micelle concentration(CMC) of the copolymer micelle was measured by flourescence technique with pyrene as probe.The size distribution of micelle was characterized on a dynamic laser light scattering particle size analyzer and its shape was observed by transmission electron microscopy(TEM).The results show that the copolymer could self-assemble into micelle with a low CMC,and the effective diameter of the micelle was 116.3 nm.The methotrexate(MTX)-loaded micelle were prepared and the drug loading content(DLC) was 22.86%.The MTX-loaded copolymer exhibited a better water-solubility than the free drug.

  5. Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs.

    Directory of Open Access Journals (Sweden)

    Alex Loukas

    2005-10-01

    Full Text Available BACKGROUND: Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. METHODS AND FINDINGS: We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056 and fecal egg counts (p = 0.018 in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049 and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood. CONCLUSION: To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine.

  6. Selective Impairment of Spatial Cognition Caused by Autoantibodies to the N-Methyl-d-Aspartate Receptor

    Directory of Open Access Journals (Sweden)

    Eric H. Chang

    2015-07-01

    Full Text Available Patients with systemic lupus erythematosus (SLE experience cognitive abnormalities in multiple domains including processing speed, executive function, and memory. Here we show that SLE patients carrying antibodies that bind DNA and the GluN2A and GluN2B subunits of the N-methyl-d-aspartate receptor (NMDAR, termed DNRAbs, displayed a selective impairment in spatial recall. Neural recordings in a mouse model of SLE, in which circulating DNRAbs penetrate the hippocampus, revealed that CA1 place cells exhibited a significant expansion in place field size. Structural analysis showed that hippocampal pyramidal cells had substantial reductions in their dendritic processes and spines. Strikingly, these abnormalities became evident at a time when DNRAbs were no longer detectable in the hippocampus. These results suggest that antibody-mediated neurocognitive impairments may be highly specific, and that spatial cognition may be particularly vulnerable to DNRAb-mediated structural and functional injury to hippocampal cells that evolves after the triggering insult is no longer present.

  7. Solid-State Synthesis, Characterization, and Biological Activity of the Bioinorganic Complex of Aspartic Acid and Arsenic Triiodide

    Directory of Open Access Journals (Sweden)

    Guo-Qing Zhong

    2013-01-01

    Full Text Available The bioinorganic complex of aspartic acid and arsenic triiodide was synthesized by a solid-state reaction at room temperature. The formula of the complex is AsI3[HOOCCH2CH(NH2COOH]2.5. The crystal structure of the complex belongs to monoclinic system with lattice parameters: a=1.0019 nm, b=1.5118 nm, c=2.1971 nm, and β=100.28°. The infrared spectra can demonstrate the complex formation between the arsenic ion and aspartic acid, and the complex may be a dimer with bridge structure. The result of primary biological test indicates that the complex possesses better biological activity for the HL-60 cells of the leukemia than arsenic triiodide.

  8. A double-suicide autopsy case of potassium poisoning by intravenous administration of potassium aspartate after intake of some psychopharmaceuticals.

    Science.gov (United States)

    Watanabe, K; Hasegawa, K; Suzuki, O

    2011-07-01

    We report a curious double-suicide autopsy case of both male and female who died of potassium poisoning by intravenous administration of concentrated potassium aspartate solution. The plasma concentrations of potassium of the male and female subjects were as high as 49.7 and 62.8 mEq/L, respectively. In addition to the high concentrations of potassium, toxic levels of phenobarbital, promethazine and chlorpromazine, and relatively low levels of etizolam and brotizolam were also detected from whole blood and urine specimens of both cadavers. Twenty empty plastic bottles (10-mL capacity) labeled 'ASPARA® Potassium Injection 10 mEq' were found at the suicide spot. To our knowledge, this is the first description for suicidal death by potassium aspartate; in all of the previous literature, they used potassium chloride intravenously or per os. PMID:20670988

  9. Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16☆

    Science.gov (United States)

    Barathy, Deivanayaga V.; Suguna, Kaza

    2013-01-01

    We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. PMID:23923105

  10. R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

    Science.gov (United States)

    Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

    2016-01-01

    The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport. PMID:26849958

  11. Single exposure to cocaine impairs aspartate uptake in the pre-frontal cortex via dopamine D1-receptor dependent mechanisms.

    Science.gov (United States)

    Sathler, Matheus Figueiredo; Stutz, Bernardo; Martins, Robertta Silva; Dos Santos Pereira, Maurício; Pecinalli, Ney Roner; Santos, Luis E; Taveira-da-Silva, Rosilane; Lowe, Jennifer; de Freitas, Isis Grigorio; de Melo Reis, Ricardo Augusto; Manhães, Alex C; Kubrusly, Regina C C

    2016-08-01

    Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC. PMID:27208619

  12. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-d-aspartate receptor subunits and d-serine ☆

    OpenAIRE

    Savignac, Helene M.; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P.E.; Tzortzis, George; Burnet, Philip W. J.

    2013-01-01

    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we exami...

  13. Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes

    OpenAIRE

    Revuelta, M.V.; Kan, van, J.; Kay, J; Have, ten, P.

    2014-01-01

    The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the fungal AP sequence space and to obtain an in-depth understanding of fungal AP evolution. Using a comprehensive phylogeny of approximately 700 AP sequences from the complete proteomes of 87 fungi and 20 ...

  14. Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda

    International Nuclear Information System (INIS)

    Crystals of the aspartate carbamoyltransferase of the psychrophile M. profunda diffract X-rays to 2.85 Å. Three catalytic and three regulatory subunits are predicted per asymmetric unit. Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the α-amino group of l-aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-l-aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 Å resolution showed that the crystal belongs to space group P3221, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 Å, α = β = 90, γ = 120°, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality

  15. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

  16. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD

    International Nuclear Information System (INIS)

    The recombinant aspartate transcarbamoylase domain of human CAD was expressed in E. coli, purified and crystallized in the presence and absence of the inhibitor PALA. X-ray diffraction data sets were collected for both crystal forms at 2.1 Å resolution. Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-l-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6322, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning

  17. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  18. Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals

    Science.gov (United States)

    Errico, F; Nisticò, R; Di Giorgio, A; Squillace, M; Vitucci, D; Galbusera, A; Piccinin, S; Mango, D; Fazio, L; Middei, S; Trizio, S; Mercuri, N B; Teule, M A; Centonze, D; Gozzi, A; Blasi, G; Bertolino, A; Usiello, A

    2014-01-01

    D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans. PMID:25072322

  19. Immobilization of Escherichia coli Cells Containing Aspartase Activity with Polyurethane and Its Application for l-Aspartic Acid Production

    Science.gov (United States)

    Fusee, Murray C.; Swann, Wayne E.; Calton, Gary J.

    1981-01-01

    Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37°C as compared to 25°C operating temperature. The pH optimum was broad, ranging from 8.5 to 9.2. Increasing the concentration of ammonium fumarate to 1.5 M from 1.0 M negatively affected the reaction rate. l-Aspartic acid was produced at an average rate of 2.18 × 10−4 mol/min per g (wet weight) of immobilized E. coli cells with a 37°C substrate solution consisting of 1.0 M ammonium fumarate with 1 mM Mg2+ (pH 9.0). PMID:16345865

  20. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

    Science.gov (United States)

    Kim, Sun-Ki; Min, Won-Ki; Park, Yong-Cheol; Seo, Jin-Ho

    2015-11-01

    Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.

  1. The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

    Energy Technology Data Exchange (ETDEWEB)

    Dostál, Ji& #345; í; Brynda, Ji& #345; í; Hrušková-Heidingsfeldová, Olga; Sieglová, Irena; Pichová, Iva; & #344; ezá& #269; ová, Pavlína; (ASCR-ICP)

    2010-09-01

    Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.

  2. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    Science.gov (United States)

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  3. An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.

    Science.gov (United States)

    Friedt, Jenna; Leavens, Fern M V; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

    2014-04-01

    Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.

  4. Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals.

    Science.gov (United States)

    Errico, F; Nisticò, R; Di Giorgio, A; Squillace, M; Vitucci, D; Galbusera, A; Piccinin, S; Mango, D; Fazio, L; Middei, S; Trizio, S; Mercuri, N B; Teule, M A; Centonze, D; Gozzi, A; Blasi, G; Bertolino, A; Usiello, A

    2014-01-01

    D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans.

  5. Measurement of aspartic acid in oilseed rape leaves under herbicide stress using near infrared spectroscopy and chemometrics.

    Science.gov (United States)

    Zhang, Chu; Kong, Wenwen; Liu, Fei; He, Yong

    2016-01-01

    Oilseed rape is used as both food and a renewable energy resource. Physiological parameters, such as the amino acid aspartic acid, can indicate the growth status of oilseed rape. Traditional detection methods are laborious, time consuming, costly, and not usable in the field. Here, we investigate near infrared spectroscopy (NIRS) as a fast and non-destructive detection method of aspartic acid in oilseed rape leaves under herbicide stress. Different spectral pre-processing methods were compared for optimal prediction performance. The variable selection methods were applied for relevant variable selection, including successive projections algorithm (SPA), Monte Carlo-uninformative variable elimination (MC-UVE) and random frog (RF). The selected effective wavelengths (EWs) were used as input by multiple linear regression (MLR), partial least squares (PLS) and least-square support vector machine (LS-SVM). The best predictive performance was achieved by SPA-LS-SVM (Raw) model using 22 EWs, and the prediction results were Rp = 0.9962 and RMSEP = 0.0339 for the prediction set. The result indicated that NIR combined with LS-SVM is a powerful new method to detect aspartic acid in oilseed rape leaves under herbicide stress. PMID:27441244

  6. Anti-N-methyl-D-aspartate receptor encephalitis: analysis of three cases

    Directory of Open Access Journals (Sweden)

    Hui SU

    2015-07-01

    Full Text Available Objective To study clinical features, diagnosis, therapy response and prognosis of anti-N-methyl-D-aspartate receptor (anti-NMDAR encephalitis.  Methods Three cases with anti-NMDAR encephalitis were reported. The clinical features, laboratory examinations, imaging, EEG and therapy response of 3 cases were retrospectively analyzed, and also related literatures were reviewed.  Results Two patients were young male and one patient was old female. Main symptoms included psychiatric symptoms in 3 cases (mania in 2 male patients and stupor in the female patient, epilepsy in 2 cases and respiratory failure in one case. The results of MRI examination revealed normal, while EEG examination showed abnormal in all cases. No tumor was detected in any of these patients. Lumbar puncture revealed normal cerebrospinal fluid (CSF pressure (3 cases, elevated white blood cell (WBC, 3 cases and protein quantification (one case. All cases were confirmed to have the disease by detection of anti-NMDAR antibodies in serum and CSF. One male patient got better after receiving immunotherapy with methylprednisolone and intravenous immunoglobulin (IVIg, but psychiatric symptoms were left over. Another male patient had no response to the above treatment. But the female patient was improved without immunotherapy. All 3 cases were followed up for one year after being discharged. One male patient died by accident because of mental disorders. Another male patient showed no sign of relief. The female patient got mild personality and memory change.  Conclusions Anti-NMDAR encephalitis is a new type of autoimmune encephalitis. It is characterized by fever, memory deficits, seizures, disturbance of consciousness, and autonomic dysfunction in males and females of all ages. This type of encephalitis is often associated with teratoma, and has a good response to immunotherapy. There is a certain correlation between progression and prognosis. DOI: 10.3969/j.issn.1672-6731.2015.07.013

  7. Selective vulnerabilities of N-methyl-D-aspartate (NMDA receptors during brain aging

    Directory of Open Access Journals (Sweden)

    Brenna L Brim

    2010-03-01

    Full Text Available N-methyl-D-aspartate (NMDA receptors are present in high density within the cerebral cortex and hippocampus and play an important role in learning and memory. NMDA receptors are negatively affected by aging, but these effects are not uniform in many different ways. This review discusses the selective age-related vulnerabilities of different binding sites of the NMDA receptor complex, different subunits that comprise the complex, and the expression and functions of the receptor within different brain regions. Spatial reference, passive avoidance, and working memory, as well as place field stability and expansion all involve NMDA receptors. Aged animals show deficiencies in these functions, as compared to young, and some studies have identified an association between age-associated changes in the expression of NMDA receptors and poor memory performance. A number of diet and drug interventions have shown potential for reversing or slowing the effects of aging on the NMDA receptor. On the other hand, there is mounting evidence that the NMDA receptors that remain within aged individuals are not always associated with good cognitive functioning. This may be due to a compensatory response of neurons to the decline in NMDA receptor expression or a change in the subunit composition of the remaining receptors. These studies suggest that developing treatments that are aimed at preventing or reversing the effects of aging on the NMDA receptor may aid in ameliorating the memory declines that are associated with aging. However, we need to be mindful of the possibility that there may also be negative consequences in aged individuals.

  8. Anti-N-methyl-D-aspartate-receptor encephalitis: diagnosis, optimal management, and challenges

    Directory of Open Access Journals (Sweden)

    Mann AP

    2014-07-01

    Full Text Available Andrea P Mann,1 Elena Grebenciucova,2 Rimas V Lukas21Department of Psychiatry and Behavioral Neuroscience, 2Department of Neurology, University of Chicago, Chicago, IL, USAObjective: Anti-N-methyl-D-aspartate-receptor (NMDA-R encephalitis is a new autoimmune disorder, often paraneoplastic in nature, presenting with complex neuropsychiatric symptoms. Diagnosed serologically, this disorder is often responsive to immunosuppressant treatment. The objective of this review is to educate clinicians on the challenges of diagnosis and management of this disorder.Materials and methods: A review of the relevant literature on clinical presentation, pathophysiology, and recommended management was conducted using a PubMed search. Examination of the results identified articles published between 2007 and 2014.Results: The literature highlights the importance of recognizing early common signs and symptoms, which include hallucinations, seizures, altered mental status, and movement disorders, often in the absence of fever. Although the presence of blood and/or cerebrospinal fluid autoantibodies confirms diagnosis, approximately 15% of patients have only positive cerebrospinal fluid titers. Antibody detection should prompt a search for an underlying teratoma or other underlying neoplasm and the initiation of first-line immunosuppressant therapy: intravenous methylprednisolone, intravenous immunoglobulin, or plasmapheresis, or a combination thereof. Second-line treatment with rituximab or cyclophosphamide should be implemented if no improvement is noted after 10 days. Complications can include behavioral problems (eg, aggression and insomnia, hypoventilation, catatonia, and autonomic instability. Those patients who can be managed outside an intensive care unit and whose tumors are identified and removed typically have better rates of remission and functional outcomes.Conclusion: There is an increasing need for clinicians of different specialties, including

  9. Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma

    Science.gov (United States)

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P.; Martin, William; De Groot, Anne S.; Gregory, Stephen H.; Wands, Jack R.

    2015-01-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA Class I and II peptides to generate antigen specific CD4+ and CD8+ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4+ and CD8+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity. PMID:25629522

  10. Serum γ-glutamyltransferase, alanine aminotransferase, and aspartate aminotransferase activity in Iranian healthy blood donor men

    Institute of Scientific and Technical Information of China (English)

    Hossein Khedmat; Nasrin Zarei; Farahnaz Fallahian; Hassan Abolghasemi; Bashir Hajibeigi; Zohre Attarchi; Farshid Alaeddini; Mohammad Taghi Holisaz; Masoumeh Pourali; Shahin Sharifi

    2007-01-01

    AIM: To determine serum γ-glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity, and to assess their correlation with demographic and clinical findings in healthy blood donors.METHODS: This cross-sectional study was performed in 934 male blood donors, aged 18 to 68 years, who consecutively attended Tehran blood transfusion service in 2006. All participants were seronegative for HBV or HCV infections, non alcohol users, and all underwent a standard interview and anthropometric tests. Clinical and biochemical parameters including AST, ALT, and GGT activities were determined. Patients taking drugs known to cause hepatic fat deposition were excluded. For AST, ALT, and GGT variables, we used 33.33 and 66.66 percentiles, so that each of them was divided into three tertiles.RESULTS: Mean AST, ALT, and GGT activities were 25.26 ± 12.58 U/L (normal range 5-35 U/L), 33.13 ± 22.98 (normal range 5-35 U/L), and 25.11 ± 18.32 (normal range 6-37 U/L), respectively. By univariate analyses, there were significant associations between increasing AST, ALT, or GGT tertiles and age, body weight, body mass index, and waist and hip circumferences (P < 0.05). By multiple linear regression analyses, ALT was found to be positively correlated with dyslipidemia (B = 6.988, P = 0.038), whereas ALT and AST were negatively correlated with age. AST, ALT, and GGT levels had positive correlation with family history of liver disease (B = 15.763, P < 0.001), (B = 32.345, P < 0.001), (B =24.415, P < 0.001), respectively.CONCLUSION: Although we did not determine the cutoffs of the upper normal limits for AST, ALT, and GGT levels, we would suggest screening asymptomatic patients with dyslipidemia and also subjects with a family history of liver disease.

  11. Proton transport properties of poly(aspartic acid) with different average molecular weights

    International Nuclear Information System (INIS)

    Research highlights: → Seven polymers with different average molecular weights were synthesized. → The proton conductivity depended on the number-average degree of polymerization. → The difference of the proton conductivities was more than one order of magnitude. → The number-average molecular weight contributed to the stability of the polymer. - Abstract: We synthesized seven partially protonated poly(aspartic acids)/sodium polyaspartates (P-Asp) with different average molecular weights to study their proton transport properties. The number-average degree of polymerization (DP) for each P-Asp was 30 (P-Asp30), 115 (P-Asp115), 140 (P-Asp140), 160 (P-Asp160), 185 (P-Asp185), 205 (P-Asp205), and 250 (P-Asp250). The proton conductivity depended on the number-average DP. The maximum and minimum proton conductivities under a relative humidity of 70% and 298 K were 1.7 . 10-3 S cm-1 (P-Asp140) and 4.6 . 10-4 S cm-1 (P-Asp250), respectively. Differential thermogravimetric analysis (TG-DTA) was carried out for each P-Asp. The results were classified into two categories. One exhibited two endothermic peaks between t = (270 and 300) oC, the other exhibited only one peak. The P-Asp group with two endothermic peaks exhibited high proton conductivity. The high proton conductivity is related to the stability of the polymer. The number-average molecular weight also contributed to the stability of the polymer.

  12. N-Methyl D-Aspartic Acid (NMDA Receptors and Depression

    Directory of Open Access Journals (Sweden)

    Enver Yusuf Sivrioglu

    2009-06-01

    Full Text Available The monoaminergic hypothesis of depression has provided the basis for extensive research into the pathophysiology of mood disorders and has been of great significance for the development of effective antidepressants. Current antidepressant treatments not only increase serotonin and/or noradrenaline bioavailability but also originate adaptive changes increasing synaptic plasticity. Novel approaches to depression and to antidepressant therapy are now focused on intracellular targets that regulate neuroplasticity and cell survival. Accumulating evidence indicates that there is an anatomical substrate for such a devastating neuropsychiatric disease as major depression. Loss of synaptic plasticity and hippocampal atrophy appear to be prominent features of this highly prevalent disorder. A combination of genetic susceptibility and environmental factors make hippocampal neurons more vulnerable to stress. Abundant experimental evidence indicates that stress causes neuronal damage in brain regions, notably in hippocampal subfields. Stress-induced activation of glutamatergic transmission may induce neuronal cell death through excessive stimulation of N-methyl-D-aspartic acid (NMDA receptors. Recent studies mention that the increase of nitric oxide synthesis and inflammation in major depression may contribute to neurotoxicity through NMDA receptor. Both standard antidepressants and NMDA receptor antagonists are able to prevent stress-induced neuronal damage. NMDA antagonists are effective in widely used animal models of depression and some of them appear to be effective also in the few clinical trials performed to date. We are still far from understanding the complex cellular and molecular events involved in mood disorders. There appears to be an emerging role for glutamate neurotransmission in the search for the pathogenesis of major depression. Attenuation of NMDA receptor function mechanism appears to be a promising target in the search for a more

  13. Sensitization of rat facial cutaneous mechanoreceptors by activation of peripheral N-methyl-d-aspartate receptors.

    Science.gov (United States)

    Gazerani, Parisa; Dong, Xudong; Wang, Mianwei; Kumar, Ujendra; Cairns, Brian E

    2010-03-10

    The effect of subcutaneous injection of glutamate on the mechanical sensitivity of rat facial cutaneous mechanoreceptors was examined. Individual facial mechanoreceptors were recorded in the trigeminal ganglion of anesthetized Sprague-Dawley rats. An electronic von Frey hair was used to measure the mechanical threshold (MT) of the afferent fibers at baseline and following subcutaneous injection of glutamate (0, 0.01, 0.1, 1M; 10microl) or glutamate (0, 0.1M) plus the competitive N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV; 0.01M). Subcutaneous injections were randomized and the investigator was unaware of their content. Changes in MT were assessed with a repeated measure ANOVA with time, sex and treatment as factors. Immunohistochemistry was used to confirm NMDA receptor expression by cutaneous nerve fibers. A total of 100 (50 per sex) facial mechanoreceptors were recorded from 61 (32 females, 29 males) rats in two separate experiments. Subcutaneous injections of higher concentrations of glutamate (1, 0.1M) induced a significant mechanical sensitization of skin afferent fibers (compared to 0 and 0.01M). Females (EC(50)=16.2mM) were more sensitive to glutamate than males (EC(50)=73.0mM). Facial cutaneous nerve fibers in both sexes expressed NMDA receptors. APV blocked the mechanical sensitization of the afferent fibers treated by glutamate 0.1M in both sexes with a lower effect in females at a 10-20minute post-injection. Subcutaneous injection of glutamate mechanically sensitizes rat facial cutaneous mechanoreceptors through activation of peripheral NMDA receptors. Peripheral NMDA receptor antagonists may be considered for craniofacial pain.

  14. Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.

    Science.gov (United States)

    Chen, Ying; Wang, Qi; Zhang, Chun; Li, Xiunan; Gao, Qiang; Dong, Changqing; Liu, Yongdong; Su, Zhiguo

    2016-06-01

    Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency. PMID:26826314

  15. An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.).

    Science.gov (United States)

    Devaraj, K B; Gowda, Lalitha R; Prakash, V

    2008-02-01

    The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500+/-500Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60+/-0.5 degrees C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly beta-structures. The purified protease is thermostable. The apparent T(m), (mid point of thermal inactivation) was found to be 70+/-0.5 degrees C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E(a)) was found to be 44.0+/-0.3kcal mol(-1). The activation enthalpy (DeltaH *), free energy change (DeltaG *) and entropy (DeltaS *) were estimated to be 43+/-4kcal mol(-1), -26+/-3kcal mol(-1) and 204+/-10cal mol(-1)K(-1), respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P1 position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.

  16. D-Aspartic acid: an endogenous amino acid with an important neuroendocrine role.

    Science.gov (United States)

    D'Aniello, Antimo

    2007-02-01

    D-Aspartic acid (d-Asp), an endogenous amino acid present in vertebrates and invertebrates, plays an important role in the neuroendocrine system, as well as in the development of the nervous system. During the embryonic stage of birds and the early postnatal life of mammals, a transient high concentration of d-Asp takes place in the brain and in the retina. d-Asp also acts as a neurotransmitter/neuromodulator. Indeed, this amino acid has been detected in synaptosomes and in synaptic vesicles, where it is released after chemical (K(+) ion, ionomycin) or electric stimuli. Furthermore, d-Asp increases cAMP in neuronal cells and is transported from the synaptic clefts to presynaptic nerve cells through a specific transporter. In the endocrine system, instead, d-Asp is involved in the regulation of hormone synthesis and release. For example, in the rat hypothalamus, it enhances gonadotropin-releasing hormone (GnRH) release and induces oxytocin and vasopressin mRNA synthesis. In the pituitary gland, it stimulates the secretion of the following hormones: prolactin (PRL), luteinizing hormone (LH), and growth hormone (GH) In the testes, it is present in Leydig cells and is involved in testosterone and progesterone release. Thus, a hypothalamus-pituitary-gonads pathway, in which d-Asp is involved, has been formulated. In conclusion, the present work is a summary of previous and current research done on the role of d-Asp in the nervous and endocrine systems of invertebrates and vertebrates, including mammals. PMID:17118457

  17. Alanine and aspartate aminotransferase and glutamine-cycling pathway: Their roles in pathogenesis of metabolic syndrome

    Institute of Scientific and Technical Information of China (English)

    Silvia Sookoian; Carlos J Pirola

    2012-01-01

    Although new research technologies are constantly used to look either for genes or biomarkers in the prediction of metabolic syndrome (MS),the pathogenesis and pathophysiology of this complex disease remains a major challenge.Interestingly,Cheng et al recently investigated possible pathways underlying MS by high-throughput metabolite profiling in two large and well characterized community-based cohorts.The authors explored by liquid chromatography and mass spectrometry the plasma concentrations of 45distinct metabolites and examined their relation to cardiometabolic risk,and observed that metabolic risk factors such as obesity,insulin resistance (IR),high blood pressure,and dyslipidemia were associated with several metabolites,including branched-chain amino acids,other hydrophobic amino acids,tryptophan breakdown products,and nucleotide metabolites.In addition,the authors found a significant association of IR traits with glutamine,glutamate and the glutamineto-glutamate ratio.These data provide new insight into the pathogenesis of MS-associated phenotypes and introduce a crucial role of glutamine-cycling pathway as prominently involved in the development of metabolic risk.We consider that the hypothesis about the role of abnormal glutamate metabolism in the pathogenesis of the MS is certainly challenging and suggests the critical role of the liver in the global metabolic modulation as glutamate metabolism is linked with aminotransferase reactions.We discuss here the critical role of the "liver metabolism" in the pathogenesis of the MS and IR,and postulate that before fatty liver develops,abnormal levels of liver enzymes,such as alanine and aspartate aminotransferases might reflect high levels of hepatic transamination of amino acids in the liver.

  18. Comments on the paper: 'Optical reflectance, optical refractive index and optical conductivity measurements of nonlinear optics for L-aspartic acid nickel chloride single crystal'

    Science.gov (United States)

    Srinivasan, Bikshandarkoil R.; Naik, Suvidha G.; Dhavskar, Kiran T.

    2016-02-01

    We argue that the 'L-aspartic acid nickel chloride' crystal reported by the authors of the title paper (Optics Communications, 291 (2013) 304-308) is actually the well-known diaqua(L-aspartato)nickel(II) hydrate crystal.

  19. Role of the Aspartate Transaminase and Platelet Ratio Index in Assessing Hepatic Fibrosis and Liver Inflammation in Adolescent Patients with HBeAg-Positive Chronic Hepatitis B

    Directory of Open Access Journals (Sweden)

    Yu Zhijian

    2015-01-01

    Full Text Available This study described an index of aspartate aminotransferase-to-platelet ratio index (APRI to assess hepatic fibrosis with limited expense and widespread availability compared to the liver biopsy in adolescent patients with CHB.

  20. Fetal and perinatal outcomes in type 1 diabetes pregnancy: a randomized study comparing insulin aspart with human insulin in 322 subjects

    DEFF Research Database (Denmark)

    Hod, Moshe; Damm, Peter; Kaaja, Risto;

    2008-01-01

    The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy.......The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy....

  1. Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate β-Decarboxylase Activity and Application to l-Alanine Production

    Science.gov (United States)

    Fusee, Murray C.; Weber, Jennifer E.

    1984-01-01

    Whole cells of Pseudomonas dacunhae containing l-aspartate β-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate β-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45°C, respectively. Immobilized P. dacunhael-aspartate β-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM α-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate β-decarboxylase activity was observed over a 31-day period. PMID:16346636

  2. Investigations on CXCL13 in Anti–N-Methyl-D-Aspartate Receptor Encephalitis

    Science.gov (United States)

    Leypoldt, Frank; Höftberger, Romana; Titulaer, Maarten J.; Armangue, Thaís; Gresa-Arribas, Nuria; Jahn, Holger; Rostásy, Kevin; Schlumberger, Wolfgang; Meyer, Thomas; Wandinger, Klaus-Peter; Rosenfeld, Myrna R.; Graus, Francesc; Dalmau, Josep

    2016-01-01

    IMPORTANCE Anti–N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe but treatable autoimmune encephalitis affecting mainly young adults and children. The lack of suitable biomarkers of disease activity makes treatment decisions and identification of relapses challenging. OBJECTIVE To determine the levels of the B-cell–attracting C-X-C motif chemokine 13 (CXCL13) in serum samples and cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis and whether they can be used as biomarkers of treatment response and outcome. DESIGN, SETTINGS, AND PARTICIPANTS Retrospective cohort study of 167 patients consecutively diagnosed as having anti-NMDAR encephalitis between May 1, 2008, and January 31, 2013. Concentration of CXCL13 was determined with enzyme-linked immunosorbent assay in all available patients’ samples (272 CSF and 55 serum samples). Samples from 25 patients with noninflammatory neurological disorders and 9 with neuroborreliosis served as controls. Expression of CXCL13 in the brain biopsy of a patient with anti-NMDAR encephalitis was determined by immunohistochemistry. MAIN OUTCOMES AND MEASURES Percentage of patients with anti-NMDAR encephalitis and elevated CXCL13 in CSF. RESULTS Compared with control individuals, 70% of patients with early-stage anti-NMDAR encephalitis had increased CXCL13 in CSF (>7 pg/mL; P 1047 pg/mL; P > .99). High concentration of CSF CXCL13 was associated with the presence of prodromal fever or headache (P = .01), limited response to therapy (P = .003), clinical relapses (P = .03), and intrathecal NMDAR-antibody synthesis (P < .001). Among patients with monophasic disease assessed 2 to 6 months after starting treatment, 10 of 15 with limited treatment response vs 0 of 13 with favorable response had increased CSF CXCL13 (specificity, 100%; 95% CI, 75–100 and sensitivity, 67%; 95% CI, 38–88; P = .02). Six of 12 patients had elevated CSF CXCL13 at relapse including 3 with previously normal levels. In brain

  3. Kinetic isotope effect studies on aspartate aminotransferase: Evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Julin, D.A.; Kirsch, J.F. (Univ. of California, Berkeley (USA))

    1989-05-02

    The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with (alpha-2H)-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H{sub 2}O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D{sub 2}O). The D{sub 2}O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D{sub 2}OV = 2.21 +/- 0.07 and D{sub 2}OV/KAsp = 1.70 +/- 0.03 with ({alpha}-1H)-L-aspartate; D{sub 2}OV = 2.34 +/- 0.12 and D{sub 2}OV/KAsp = 1.82 +/- 0.06 with ({alpha}-2H)-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H{sub 2}O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D{sub 2}O) coupled with the relatively small SKIEs (D{sub 2}OV = 1.58 +/- 0.04 and D{sub 2}OV/KGlu = 1.25 +/- 0.05 with ({alpha}-1H)-L-glutamate; D{sub 2}OV = 1.46 +/- 0.06 and D{sub 2}OV/KGlu = 1.16 +/- 0.05 with (alpha-2H)-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.

  4. Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

    Science.gov (United States)

    Xu, Xinyi; Gu, Liping; He, Ping; Zhou, Ruanbao

    2015-06-01

    Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N(2)) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N(2)-fixing cyanobacteria. In this report, five putative AspAT genes (alr1039, all2340, alr2765, all4327 and alr4853) were identified in the N(2)-fixing heterocystous cyanobacterium Anabaena sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H(6)) were overexpressed in Escherichia coli and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity in vitro using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed in vitro. Alr4853-H(6) showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and alr4853 was further identified to be a Fox gene (requiring fixed N(2) for growth in the presence of oxygen). Four out of five P(aspAT)-gfp transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that alr4853 expression was increased by 2.3-fold after 24 h of N(2) deprivation. Taken together, these findings add to our understanding of the role of AspATs in N(2)-fixing within heterocystous cyanobacteria.

  5. Aspartate Rescues S-phase Arrest Caused by Suppression of Glutamine Utilization in KRas-driven Cancer Cells.

    Science.gov (United States)

    Patel, Deven; Menon, Deepak; Bernfeld, Elyssa; Mroz, Victoria; Kalan, Sampada; Loayza, Diego; Foster, David A

    2016-04-22

    During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

  6. The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

    Science.gov (United States)

    Denu, J M; Zhou, G; Guo, Y; Dixon, J E

    1995-03-14

    The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Psychotic symptoms in anti-N-methyl-d-aspartate (NMDA) receptor encephalitis: A case report and challenges.

    Science.gov (United States)

    Sharma, Pawan; Sagar, Rajesh; Patra, Bichitrananda; Saini, Lokesh; Gulati, Sheffali; Chakrabarty, Biswaroop

    2016-08-01

    Anti-N-methyl-d-aspartate (NMDA) receptor encephalitis, only recently first described, is an increasingly well-recognized inflammatory encephalitis that is seen in children and adults. An 11-year old girl admitted to the psychiatry ward with a presentation of acute psychosis was diagnosed with NMDA receptor encephalitis following neurology referral and was treated accordingly. This case highlights psychiatric manifestations in encephalitis and the need for the psychiatrist to have high index of suspicion when atypical symptoms (e.g., dyskinesia, seizure, fever etc.) present in acutely psychotic patients. PMID:27520914

  8. Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

    OpenAIRE

    Wallace, B; Yang, Y. J.; Hong, J S; Lum, D

    1990-01-01

    A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter co...

  9. D-Aspartate Modulates Nociceptive-Specific Neuron Activity and Pain Threshold in Inflammatory and Neuropathic Pain Condition in Mice

    Directory of Open Access Journals (Sweden)

    Serena Boccella

    2015-01-01

    Full Text Available D-Aspartate (D-Asp is a free D-amino acid found in the mammalian brain with a temporal-dependent concentration based on the postnatal expression of its metabolizing enzyme D-aspartate oxidase (DDO. D-Asp acts as an agonist on NMDA receptors (NMDARs. Accordingly, high levels of D-Asp in knockout mice for Ddo gene (Ddo−/− or in mice treated with D-Asp increase NMDAR-dependent processes. We have here evaluated in Ddo−/− mice the effect of high levels of free D-Asp on the long-term plastic changes along the nociceptive pathway occurring in chronic and acute pain condition. We found that Ddo−/− mice show an increased evoked activity of the nociceptive specific (NS neurons of the dorsal horn of the spinal cord (L4–L6 and a significant decrease of mechanical and thermal thresholds, as compared to control mice. Moreover, Ddo gene deletion exacerbated the nocifensive responses in the formalin test and slightly reduced pain thresholds in neuropathic mice up to 7 days after chronic constriction injury. These findings suggest that the NMDAR agonist, D-Asp, may play a role in the regulation of NS neuron electrophysiological activity and behavioral responses in physiological and pathological pain conditions.

  10. D-Aspartate Modulates Nociceptive-Specific Neuron Activity and Pain Threshold in Inflammatory and Neuropathic Pain Condition in Mice

    Science.gov (United States)

    Boccella, Serena; Vacca, Valentina; Errico, Francesco; Marinelli, Sara; Squillace, Marta; Di Maio, Anna; Vitucci, Daniela; Palazzo, Enza; De Novellis, Vito; Maione, Sabatino; Pavone, Flaminia; Usiello, Alessandro

    2015-01-01

    D-Aspartate (D-Asp) is a free D-amino acid found in the mammalian brain with a temporal-dependent concentration based on the postnatal expression of its metabolizing enzyme D-aspartate oxidase (DDO). D-Asp acts as an agonist on NMDA receptors (NMDARs). Accordingly, high levels of D-Asp in knockout mice for Ddo gene (Ddo−/−) or in mice treated with D-Asp increase NMDAR-dependent processes. We have here evaluated in Ddo−/− mice the effect of high levels of free D-Asp on the long-term plastic changes along the nociceptive pathway occurring in chronic and acute pain condition. We found that Ddo−/− mice show an increased evoked activity of the nociceptive specific (NS) neurons of the dorsal horn of the spinal cord (L4–L6) and a significant decrease of mechanical and thermal thresholds, as compared to control mice. Moreover, Ddo gene deletion exacerbated the nocifensive responses in the formalin test and slightly reduced pain thresholds in neuropathic mice up to 7 days after chronic constriction injury. These findings suggest that the NMDAR agonist, D-Asp, may play a role in the regulation of NS neuron electrophysiological activity and behavioral responses in physiological and pathological pain conditions. PMID:25629055

  11. ASPARTATE TRANSAMINASE - IS IT USEFUL AS A BIOCHEMICAL MARKER AND AS A PREDICTOR OF SEVERITY OF PREGNANCY-INDUCED HYPERTENSION AND ITS COMPLICATION

    Directory of Open Access Journals (Sweden)

    Rupinder

    2016-03-01

    Full Text Available OBJECTIVES To compare serum Aspartate Transaminase of normotensive pregnant women with those of pre-eclamptic and eclamptic women. To determine the relationship of levels of serum Aspartate Transaminase with severity of pregnancy-induced hypertension and its complications. METHOD The study was carried out on pregnant hypertensive patients attending Outpatient Department of Obstetrics and Gynaecology Department, AMCH Dibrugarh, Assam from 1 st July 2013 to 30 th June 2014. Normotensive pregnant women were taken as controls. Each serum sample from the control group as well as study group was estimated for Aspartate Transaminase using standard methods, and a comparison is drawn and analysed using t-test and chi-square test. RESULTS Serum Aspartate Transaminase levels were high in the study group. The levels of this enzyme were normal in the control group. CONCLUSION Aspartate Transaminase levels in patients suffering from preeclampsia and its complications are consistently higher compared to the normotensive pregnant patients. To determine the usefulness of inclusion of this enzyme along with other cardiac enzymes in the panel of investigations of pregnant women universally needs further large scale comparative studies.

  12. Counter-regulatory hormone responses to spontaneous hypoglycaemia during treatment with insulin Aspart or human soluble insulin. A double-blinded randomised cross-over study

    DEFF Research Database (Denmark)

    Brock-Jacobsen, Iben; Vind, B F; Korsholm, L;

    2011-01-01

    To compare insulin Aspart and human insulin with respect to glycaemic control, hypoglycaemic frequency and counter-regulatory responses to spontaneous hypoglycaemia. Methods: Glycaemic control, hypoglycaemic frequency, p-insulin concentrations, insulin dosages and patients’ satisfaction were...... examined in a randomized, double-blinded cross-over study for two periods of 8 weeks. Sixteen patients with type 1 diabetes were subjected to three daily injections of human soluble insulin or Aspart in addition to Neutral Protamine Hagedorn (NPH) insulin twice daily. Each intervention period was followed...... by hospitalization where episodes of spontaneous hypoglycaemia and counter-regulatory hormone responses were evaluated from frequently obtained blood samples. Results: No difference between soluble insulin and insulin Aspart was found regarding HbA1c (7.0 0.2 vs. 7.0 0.2%, ns), hypoglycaemic...

  13. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    Directory of Open Access Journals (Sweden)

    Kazuma Ogawa

    Full Text Available (68Ga (T 1/2 = 68 min, a generator-produced nuclide has great potential as a radionuclide for clinical positron emission tomography (PET. Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Aspn (n = 2, 5, 8, 11, or 14 with easy-to-handle (67Ga, with the previously described (67Ga-DOTA complex conjugated bisphosphonate, (67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Aspn by a Fmoc-based solid-phase method, complexes were formed with (67Ga, resulting in (67Ga-DOTA-(Aspn with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67Ga-DOTA-(Aspn increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67Ga-DOTA-(Asp8, (67Ga-DOTA-(Asp11, and (67Ga-DOTA-(Asp14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67Ga-DOTA-(Aspn was lower than that of (67Ga-DOTA-Bn-SCN-HBP, blood clearance of (67Ga-DOTA-(Aspn was more rapid. Accordingly, the bone/blood ratios of (67Ga-DOTA-(Asp11 and (67Ga-DOTA-(Asp14 were comparable with those of (67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

  14. Subcellular distribution of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells within subventricular zone of adult rats

    Institute of Scientific and Technical Information of China (English)

    Zhining Li; Wenlong Lü; Hongyan Dong; Hongbin Fan; Ruiguo Dong; Tiejun Xu

    2011-01-01

    The subcellular localization of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells of the subventricular zone of adult rats was detected using electron microscopy, following immunohistochemistry and immunogold-silver double staining. Results confirmed the presence of neural stem cells in the subventricular zone, which is a key neurogenic region in the central nervous system of adult mammals. The expression of N-methyl-D-aspartic acid receptor subunit 1 was higher than that of nestin and mainly distributed in the cell membrane, cytoplasm, rough endoplasmic reticulum and Golgi complex of neural stem cells.

  15. The N-methyl-D-aspartate receptor antagonist dextromethorphan selectively reduces temporal summation of second pain in man.

    Science.gov (United States)

    Price, D D; Mao, J; Frenk, H; Mayer, D J

    1994-11-01

    Oral doses of dextromethorphan (DM), a common cough suppressant and N-methyl-D-aspartate (NMDA) receptor antagonist, and their vehicle control were given on a double-blind basis to normal volunteer human subjects who rated intensities of first and second pain in response to repeated painful electric shocks and repeated 52 degrees C heat pulses. Doses of 30 and 45 mg, but not 15 mg, were effective in attenuating temporal summation of second pain, a psychophysical correlate of temporal summation of C afferent-mediated responses of dorsal horn nociceptive neurons, termed 'wind-up'. By contrast, neither first nor second pain evoked by the first stimulus in a train of stimuli were affected by any of these doses of DM. These results further confirm temporal summation of second pain as a psychophysical correlate of wind-up by providing evidence that DM selectively reduces temporal summation of second pain, as has been shown for wind-up. PMID:7892014

  16. Mismatch responses and deviance detection in N-methyl-D-aspartate (NMDA) receptor hypofunction and developmental models of schizophrenia.

    Science.gov (United States)

    Harms, Lauren

    2016-04-01

    Reductions in the size of the mismatch negativity (MMN), an event-related potential component elicited in response to unexpected stimuli, are arguably the most robust neurophysiological findings in schizophrenia. Several studies have now demonstrated that 'true' human-like deviance detection mismatch responses (MMRs) can be generated in the rodent brain and therefore that animal models can be used to examine the neurobiology of schizophrenia-like MMR impairments and investigate the efficacy of new treatments in addressing underlying neurobiological mechanisms. Two broad categories of animal models have been examined for schizophrenia-like MMRs: models involving N-methyl-D-aspartate receptor hypofunction, and models involving an insult or exposure during development. While these models have been shown to exhibit reductions in MMRs, it is still unclear whether or not these reductions involve changes to neural adaptation to repetitive stimuli or whether they reflect impairments in the response to unexpected deviations in regular patterns. PMID:26159809

  17. Effect of heavy metals and phenol on bacterial decolourisation and COD reduction of sucrose-aspartic acid Maillard product

    Institute of Scientific and Technical Information of China (English)

    Sangeeta Yadav; Ram Chandra

    2013-01-01

    Melanodins are amino-carbonyl complex,predominantly present in sugarcane molasses based distillery wastewater as major source of colourant.The microbial decolourisation of melanoidin is a challenge due to its binding property with other co-pollutants of distillery waste.Results revealed that the presence of Zn2+ (2.00-20.00 mg/L) in melanoidin solution (1200 mg/L) stimulated the bacterial growth and sucrose-aspartic acid Maillard product (SAA) decolourisation as compared to control,while Fe3+ and Mn2+ at the same concentration inhibited the process.However,the presence of phenol (100 mg/L) along with Zn2+,Fe3+ and Mn2+ suppressed the bacterial growth,SAA decolourisation and MnP activity.The shrinkage and reduced number of bacterial cell count at higher concentration of heavy metals in presence of phenol was also observed under scanning electron microscope.

  18. Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA.

    Science.gov (United States)

    Ruiz-Ramos, Alba; Velázquez-Campoy, Adrián; Grande-García, Araceli; Moreno-Morcillo, María; Ramón-Maiques, Santiago

    2016-07-01

    CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands.

  19. Chlorogenic acid increased 5-hydroxymethylfurfural formation when heating fructose alone or with aspartic acid at two pH levels.

    Science.gov (United States)

    Zhang, Zhenhua; Zou, Yueyu; Wu, Taigang; Huang, Caihuan; Pei, Kehan; Zhang, Guangwen; Lin, Xiaohua; Bai, Weibin; Ou, Shiyi

    2016-01-01

    Chlorogenic acid (CGA) is a phenolic acid that ubiquitously exists in fruits. This work aims to investigate whether and how CGA influences HMF formation during heating fructose alone, or with an amino acid. The results showed that that CGA increased 5-hydroxymethylfurfural (HMF) formation. At pH 5.5 and 7.0, the addition of 5.0 μmol/ml CGA increased HMF formation by 49.4% and 25.2%, respectively when heating fructose alone, and by 9.0% and 16.7%, respectively when heating fructose with aspartic acid. CGA significantly increased HMF formation by promoting 3-deoxosone formation, and its conversion to HMF by inhibiting HMF elimination, especially in the Maillard reaction system. A comparison of the catalytic capacity of CGA with its six analogous compounds showed that both its di-hydroxyphenyl and carboxyl groups function in increasing HMF formation. PMID:26213045

  20. Synthesis, characterization and application of a novel chemical sand-fixing agent-poly(aspartic acid) and its composites

    Energy Technology Data Exchange (ETDEWEB)

    Yang Jun [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China); Wang Fang [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China); Fang Li [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China); Tan Tianwei [Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029 (China)]. E-mail: twtan@mail.buct.edu.cn

    2007-09-15

    A novel sand-fixing agent-poly(aspartic acid) and its composites were synthesized to improve sand particles compressive strength and anti-wind erosion properties. The relationship between the concentration of sand-fixing agent and the sand-fixing properties was studied by three kinds of aging tests. Some composites were choose to improve the sand-fixing property and the composition of 40% xanthan gum and 60% ethyl cellulose were chosen to compare sand-fixing property with lignosulfonate. The results showed that the sand-fixing and water-retaining properties of xanthan gum and ethyl cellulose composites were better than that of lignosulfonate. The biodegradability experiment showed that the PASP and its composites were environment-friendly products and the field test showed that the PASP composites could improve wind erosion disturbance. - A novel biodegradability polymer significantly improved sand particles' compressive strength and anti-wind erosion properties.

  1. Synthesis, characterization and application of a novel chemical sand-fixing agent-poly(aspartic acid) and its composites

    International Nuclear Information System (INIS)

    A novel sand-fixing agent-poly(aspartic acid) and its composites were synthesized to improve sand particles compressive strength and anti-wind erosion properties. The relationship between the concentration of sand-fixing agent and the sand-fixing properties was studied by three kinds of aging tests. Some composites were choose to improve the sand-fixing property and the composition of 40% xanthan gum and 60% ethyl cellulose were chosen to compare sand-fixing property with lignosulfonate. The results showed that the sand-fixing and water-retaining properties of xanthan gum and ethyl cellulose composites were better than that of lignosulfonate. The biodegradability experiment showed that the PASP and its composites were environment-friendly products and the field test showed that the PASP composites could improve wind erosion disturbance. - A novel biodegradability polymer significantly improved sand particles' compressive strength and anti-wind erosion properties

  2. Kainate-enhanced release of D-(3H)aspartate from cerebral cortex and striatum: reversal by baclofen and pentobarbital

    Energy Technology Data Exchange (ETDEWEB)

    Potashner, S.J.; Gerard, D.

    1983-06-01

    A study was made of the actions of the excitant neurotoxin, kainic acid, on the uptake and the release of D-(2,3-3H)aspartate (D-ASP) in slices of guinea pig cerebral neocortex and striatum. The slices took up D-ASP, reaching concentrations of the amino acid in the tissue which were 14-23 times that in the medium. Subsequently, electrical stimulation of the slices evoked a Ca2+-dependent release of a portion of the D-ASP. Kainic acid (10(-5)-10(-3) M) produced a dose-dependent inhibition of D-ASP uptake. The electrically evoked release of D-ASP was increased 1.6-2.0 fold by 10(-5) and 10(-4)M kainic acid. The kainate-enlarged release was Ca2+-dependent. Dihydrokainic acid, an analogue of kainic acid with little excitatory or toxic action, did not increase D-ASP release but depressed D-ASP uptake. Attempts were made to block the action of kainic acid with baclofen and pentobarbital, compounds which depress the electrically evoked release of L-glutamate (L-GLU) and L-aspartate (L-ASP). Baclofen (4 X 10(-6)M), an antispastic drug, and pentobarbital (10(-4)M), an anesthetic agent, each inhibited the electrically evoked release of D-ASP and prevented the enhancement of the release above control levels usually produced by 10(-4)M kainic acid. It is proposed that 10(-5) and 10(-4)M kainic acid may enhance the synaptic release of L-GLU and L-ASP from neurons which use these amino acids as transmitters. This action is prevented by baclofen and pentobarbital. In view of the possibility that cell death in Huntington's disease could involve excessive depolarization of striatal and other cells by glutamate, baclofen might be effective in delaying the loss of neurons associated with this condition.

  3. Aspartic acid racemization rate in narwhal (Monodon monoceros eye lens nuclei estimated by counting of growth layers in tusks

    Directory of Open Access Journals (Sweden)

    Eva Garde

    2012-11-01

    Full Text Available Ages of marine mammals have traditionally been estimated by counting dentinal growth layers in teeth. However, this method is difficult to use on narwhals (Monodon monoceros because of their special tooth structures. Alternative methods are therefore needed. The aspartic acid racemization (AAR technique has been used in age estimation studies of cetaceans, including narwhals. The purpose of this study was to estimate a species-specific racemization rate for narwhals by regressing aspartic acid d/l ratios in eye lens nuclei against growth layer groups in tusks (n=9. Two racemization rates were estimated: one by linear regression (r2=0.98 based on the assumption that age was known without error, and one based on a bootstrap study, taking into account the uncertainty in the age estimation (r2 between 0.88 and 0.98. The two estimated 2kAsp values were identical up to two significant figures. The 2k Asp value from the bootstrap study was found to be 0.00229±0.000089 SE, which corresponds to a racemization rate of 0.00114−yr±0.000044 SE. The intercept of 0.0580±0.00185 SE corresponds to twice the (d/l0 value, which is then 0.0290±0.00093 SE. We propose that this species-specific racemization rate and (d/l0 value be used in future AAR age estimation studies of narwhals, but also recommend the collection of tusks and eyes of narwhals for further improving the (d/l0 and 2kAsp estimates obtained in this study.

  4. Effect of heavy metals (Cu, Cd and Pb) on aspartate and alanine aminotransferase in Ruditapes philippinarum (Mollusca: Bivalvia)

    Energy Technology Data Exchange (ETDEWEB)

    Blasco, J.; Puppo, J. [Instituto de Ciencias Marinas de Andalucia, Campus Univ. Rio S. Pedro, 11510 Puerto Real, Cadiz (Spain)

    1999-02-01

    The accumulation of cadmium, copper and lead and their effects on aspartate and alanine aminotransferases in digestive gland, gills, foot and soft body in the clam Ruditapes philippinarum were examined. The animals were exposed to different concentrations: Cd (200-600 {mu}g{center_dot}l{sup -1}), Pb (350-700 {mu}g{center_dot}l{sup -1}) and Cu (10-20 {mu}g{center_dot}l{sup -1}) for 7 days. The highest concentrations were found in digestive gland for cadmium and copper, and in gills for lead, and the lowest values were observed in the foot. Aspartate aminotransferase activity (AST), in general, was not inhibited by cadmium, lead or copper during the exposure. Only in clams exposed to cadmium (600 {mu}g{center_dot}l{sup -1}, 7 days) and copper (20 {mu}g{center_dot}l{sup -1}, 5 days) were observed significant differences (P<0.05) in foot and gills, respectively, with respect to control. In the case of alanine aminotransferase activity (ALT), significant differences were observed for cadmium and lead in treated animals with respect to control. With regard to copper, a decrease in ALT was observed in gills and foot exposed to 20 {mu}g{center_dot}l{sup -1}. A significant correlation (P<0.05) was observed between ALT and metal accumulation for cadmium, copper and lead in gills. In the case of soft body, only cadmium and lead showed a significant correlation. In summary, R. philippinarum can be considered a bioindicator species for cadmium and lead accumulation and ALT could be useful as biomarker of sublethal stress for these metals in soft tissues and gills. Only gills can be considered an adequate target tissue for copper. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  5. Endopeptidase-Mediated Beta Lactam Tolerance

    OpenAIRE

    Dörr, Tobias; Davis, Brigid M.; Waldor, Matthew K.

    2015-01-01

    In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting ...

  6. Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H;

    1980-01-01

    Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl...

  7. Propofol effectively inhibits lithium-pilocarpine-induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Institute of Scientific and Technical Information of China (English)

    Henglin Wang; Zhuoqiang Wang; Weidong Mi; Cong Zhao; Yanqin Liu; Yongan Wang; Haipeng Sun

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory ef-fects of propofol on status epilepticus in rats were judged based on observation of behavior, elec-troencephalography and 24-hour survival rate. Propofol (12.5-100 mg/kg) improved status epilep-ticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly in-creased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with down-regulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.

  8. Fetal and perinatal outcomes in type 1 diabetes pregnancy : a randomized study comparing insulin aspart with human insulin in 322 subjects

    NARCIS (Netherlands)

    Hod, Moshe; Damm, Peter; Kaaja, Risto; Visser, Gerard H. A.; Dunne, Fidelma; Demidova, Irina; Hansen, Anne-Sofie Pade; Mersebach, Henriette

    2008-01-01

    OBJECTIVE: The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy. STUDY DESIGN: This was a randomized, parallel, open-label, controlled, mult

  9. Lower "N"-Acetyl-Aspartate Levels in Prefrontal Cortices in Pediatric Bipolar Disorder: A (Superscript 1]H Magnetic Resonance Spectroscopy Study

    Science.gov (United States)

    Caetano, Sheila C.; Olvera, Rene L.; Hatch, John P.; Sanches, Marsal; Chen, Hua Hsuan; Nicoletti, Mark; Stanley, Jeffrey A.; Fonseca, Manoela; Hunter, Kristina; Lafer, Beny; Pliszka, Steven R.; Soares, Jair C.

    2011-01-01

    Objective: The few studies applying single-voxel [superscript 1]H spectroscopy in children and adolescents with bipolar disorder (BD) have reported low "N"-acetyl-aspartate (NAA) levels in the dorsolateral prefrontal cortex (DLPFC), and high myo-inositol/phosphocreatine plus creatine (PCr+Cr) ratios in the anterior cingulate. The aim of this study…

  10. Stimulus intensity, cell excitation and the N-methyl-D-aspartate receptor component of sensory responses in the rat spinal cord in vivo.

    Science.gov (United States)

    Chizh, B A; Cumberbatch, M J; Herrero, J F; Stirk, G C; Headley, P M

    1997-09-01

    The importance of receptors for N-methyl-D-aspartate in synaptic plasticity and in triggering long-term pronociceptive changes is explained by their voltage-dependence. This suggests that their contribution to acute nociceptive responses would be determined both by the magnitude of synaptic input and by the level of background excitation. We have now examined the role of N-methyl-D-aspartate receptors in acute nociceptive transmission in the spinal cord. Drugs selectively affecting activity mediated by these receptors were tested on responses of dorsal horn neurons to noxious stimuli of different intensities and at different levels of ongoing spike discharge. The drugs used were the N-methyl-D-aspartate receptor channel blocker ketamine; the competitive antagonists, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (D-CPP) and D-2-amino-5-phosphonopentanoic acid (D-AP5), and the positive modulator thyrotropin-releasing hormone. The activity of dorsal horn wide dynamic range neurons was recorded extracellularly in alpha-chloralose-anaesthetized spinalized rats. Their responses to noxious stimuli (pinch, heat and electrical) were monitored in parallel with responses to iontophoretic N-methyl-D-aspartate and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Drugs were given i.v. or (D-AP5) iontophoretically. At doses that selectively inhibited responses to exogenous N-methyl-D-aspartate, ketamine (4 or 8, mean 5 mg/kg i.v.) reduced the nociceptive responses of the majority of the cells in deep dorsal horn. Ketamine also reduced wind-up of the responses to repetitive electrical stimulation. Ketamine (4 or 8 mg/kg). D-CPP (2 mg/kg), D-AP5 (iontophoretically) and thyrotrophin-releasing hormone (1 mg/kg) were tested on different magnitude nociceptive responses evoked by alternating intensities of noxious heat or pinch. In percentage terms, the less vigorous responses were affected by all four drugs as much as or more than the more vigorous

  11. Accumulation of aspartic acid421- and glutamic acid391-cleaved tau in neurofibrillary tangles correlates with progression in Alzheimer disease.

    Science.gov (United States)

    Basurto-Islas, Gustavo; Luna-Muñoz, Jose; Guillozet-Bongaarts, Angela L; Binder, Lester I; Mena, Raul; García-Sierra, Francisco

    2008-05-01

    Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E epsilon4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.

  12. Theoretical Study on the Activity of α-COOH and β-COOH of N-Phosphoryl Aspartic Acids

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between α-COOH and β-COOH in phosphoryl aspartic acid was studied by theoretical study (Hartree-Fock and Density Functional methods) in this paper. The intermediates Ⅱ containing five-membered ring were more stable than Ⅲ with six-membered ring. While for intermediates Ⅲ, the isomers with six-membered ring in apical-equatorial spanning arrangement were more stable than those with di-equatorial spanning arrangement. At B3LYP/6-31G** level, it was shown that transition states Ⅳ and V involving α-COOH or β-COOH group had energy barriers of ΔE = 58.67 kJ·mol-1 and 103.94 kJ·mol-1, respectively. These results were in agreement with the experimental data. So the α-COOH group was involved in form of the intramolecular penta-coordinate phosphoric-carboxylic mixed anhydride intermediates, but not β-COOH group.

  13. Relationship between changes of N-methyl-D-aspartate receptor activity and brain edema after brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To investigate the relationship between the changes of N-methyl-D-aspartate (NMDA) receptor activity and brain edema after injury in rats.   Methods: The brain injury models were made by using a free-falling body. The treatment model was induced by means of injecting AP5 into lateral ventricle before brain injury; water contents in brain cortex were measured with dry-wet method; and NMDA receptor activity was detected with a radio ligand binding assay.   Results: The water contents began to increase at 30 minutes and reached the peak at 6 hours after brain injury. The maximal binding (Bmax) of NMDA receptor increased significantly at 15 minutes and reached the peak at 30 minutes, then decreased gradually and had the lowest value 6 hours after brain injury. Followed the treatment with AP5, NMDA receptor activity in the injured brain showed a normal value; and the water contents were lower than that of AP5-free injury group 24 hours after brain injury.   Conclusions: It suggests that excessive activation of NMDA receptor may be one of the most important factors to induce the secondary cerebral impairments, and AP5 may protect the brain from edema after brain injury.

  14. Cysteinyl leukotriene receptor 1 is involved in /N-methyl-D-aspartate-mediated neuronal injury in mice

    Institute of Scientific and Technical Information of China (English)

    Qian DING; Er-qing WEI; Yan-jun ZHANG; Wei-ping ZHANG; Zhong CHEN

    2006-01-01

    Aim: To determine whether cysteinyl leukotriene receptor 1 (CysLT1 receptor) is involved in N-methyl-D-aspartate (NMDA)-induced excitotoxic injury in the mouse brain. Methods: Brain injury was induced by NMDA microinjection (50-150 nmol in 0.5 μL) into the cerebral cortex. The changes in CysLT1 receptor expression 24 h after NMDA injection and the effects of a CysLT1 receptor antagonist, pranlukast (0.01 and 0.1 mg/kg), an NMDA receptor antagonist, ketamine (30 mg/kg), and an antioxidant, edaravone (9 mg/kg) were observed. Results: In the NMDA-injured brain, the CysLT1 receptor mRNA, and protein expression were upregulated, and the receptor was mainly localized in the neurons and not in the astrocytes. Pranlukast, ketamine and edaravone decreased NMDA-induced injury;pranlukast (0.1 mg/kg) and ketamine inhibited the upregulated expression of the CysLT1 receptor. Conclusion: CysLT1 receptor expression in neurons is upregulated after NMDA injection, and NMDA-induced responses are inhibited by CysLT1 receptor antagonists, indicating that the increased CysLT1 receptor is involved in NMDA excitotoxicity.

  15. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536.

    Science.gov (United States)

    Dou, Kai; Wang, Zhiying; Zhang, Rongshu; Wang, Na; Fan, Haijuan; Diao, Guiping; Liu, Zhihua

    2014-12-01

    Proteases secreted by fungi belonging to the genus Trichoderma play important roles in biocontrol. In this study, the coding sequence and promoter region of the novel aspartic protease gene Asp55 were cloned from strain Trichoderma asperellum ACCC30536. Many cis-elements involved in phytopathogenic and environmental stress responses were identified in the Asp55 promoter region and may be recognized by MYB or WRKY transcription factors. The expression pattern of Asp55 under eight culture conditions was investigated by RT-qPCR. The expression level of Asp55 was up-regulated by poplar stem powder, Alternaria alternata cell wall fragments and A. alternata fermentation liquid, while it was down-regulated by carbon and nitrogen source starvation, and by powdered poplar leaves and roots. Additionally, the expression patterns of 15 genes encoding MYB transcription factors (Myb1 to Myb15) were also analyzed by RT-qPCR. Myb2 showed the most similar expression pattern with Asp55. The cDNA of Asp55 was expressed in Escherichia coli BL21, and recombinant ASP55 (rASP55) was purified. The purified rASP55 was evaluated for enzymatic activity and showed inhibitory effect on phytopathogenic A. alternata.

  16. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs

    KAUST Repository

    Alam, Tanvir

    2014-05-29

    LD motifs (leucine-aspartic acidmotifs) are short helical protein-protein interaction motifs that have emerged as key players in connecting cell adhesion with cell motility and survival. LD motifs are required for embryogenesis, wound healing and the evolution of multicellularity. LD motifs also play roles in disease, such as in cancer metastasis or viral infection. First described in the paxillin family of scaffolding proteins, LD motifs and similar acidic LXXLL interaction motifs have been discovered in several other proteins, whereas 16 proteins have been reported to contain LDBDs (LD motif-binding domains). Collectively, structural and functional analyses have revealed a surprising multivalency in LD motif interactions and a wide diversity in LDBD architectures. In the present review, we summarize the molecular basis for function, regulation and selectivity of LD motif interactions that has emerged from more than a decade of research. This overview highlights the intricate multi-level regulation and the inherently noisy and heterogeneous nature of signalling through short protein-protein interaction motifs. © 2014 Biochemical Society.

  17. Effect of developmental lead exposure on synaptic plasticity and N—methyl—D—aspartate receptor subunit in rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    RuanDY; SuiL

    2002-01-01

    Chronic lead(Pb) exposure is known to be associated with learning and memory,and cognitive dysfunction in children.Previous studies have demonstrated that Pb exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N-methyl-D-aspartate (NMDA) receptors(NMDARs).The studies described here were carried out to investigate effect of developmental Pb exposure on long-term potentiation(LTP),long-tern depression(LTD) and NMDAs subunits in rat hippocampus.The results are listed as follows:(1)low-level Pb exposture can impair the induction and maintenance of LTP in vivo and in vitro;(2)the Pb-induced impairment of LTD magnitude was an age-related decline in area CA1 of rat hippocampus;(3)chronic Pb exposure affected two components,voltage-gated calcium channel-dependent LTD and NMDARs-dependent LTD,of LTD induction in area CA1 of rat hippocampus;(4)different effects of developmental Pb exposure on NMDA receptor NR1,NR2A,NR2B,NR2C,NR2D and NR3A subunits in area CA1,CA2,CA3 and CA4 of rat hippocampus were observed;(5)the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors enriched in area CA1,CA3 and dentate gyrus and kainite receptors enriched in area CA1 and dentate gyrus of rat hippocampus were impaired by Pb exposure.

  18. IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.

    Directory of Open Access Journals (Sweden)

    Hideaki Ando

    Full Text Available Phosphatidylinositol phosphate kinases (PIPKs are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5P2, a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα and type IIα (PIPKIIα in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5P2.

  19. Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues; Souto, Tatiane Beltramini; de Oliveira, Tássio Brito; de Oliveira, Lilian Caroline Gonçalves; Karcher, Daniel; Juliano, Maria Aparecida; Juliano, Luiz; de Oliveira, Arthur H C; Rodrigues, André; Rosa, Jose C; Cabral, Hamilton

    2016-08-01

    In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese. PMID:27165660

  20. Relationship of creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and proteinuria to cardiomyopathy in the owl monkey (Aotus vociferans)

    Energy Technology Data Exchange (ETDEWEB)

    Gozalo, Alfonso S.; Chavera, Alfonso; Montoya, Enrique J.; Takano, Juan; Weller, Richard E.

    2008-02-01

    The purpose of this study was to determine serum reference values for crea- tine kinase (CK), aspartate aminotransferase (AST), and lactate dehydroge- nase (LDH) in captive-born and wild-caught owl monkeys to assess their usefulness for diagnosing myocardial disease. Urine samples were also collected and semi-quantitative tests performed. There was no statistically significant difference between CK, AST, and LDH when comparing both groups. However, when comparing monkeys with proteinuria to those without proteinuria, a statistically significant difference in CK value was observed (P = 0.021). In addition, the CK/AST ratio revealed that 29% of the animals included in this study had values suggesting cardiac infarction. Grossly, cardiac concentric hypertrophy of the left ventricle and small, pitted kidneys were the most common findings. Microscopically, myocardial fibrosis, contraction band necrosis, hypertrophy and hyperplasia of coronary arteries, medium-sized renal arteries, and afferent glomerular arteriolae were the most significant lesions, along with increased mesangial matrix and hypercellularity of glomeruli, Bowman’s capsule, and peritubular space fibroplasia. These findings suggest that CK, AST, and LDH along with urinalysis provide a reliable method for diagnosing cardiomyopathies in the owl monkey. In addition, CK/AST ratio, proteinuria, and the observed histological and ultrastructural changes suggest that Aotus vociferans suffer from arterial hypertension and chronic myocardial infarction.

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

    Directory of Open Access Journals (Sweden)

    Ohgi Takahashi

    2015-01-01

    Full Text Available 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.

  2. Single nanoparticle tracking of [Formula: see text]-methyl-d-aspartate receptors in cultured and intact brain tissue.

    Science.gov (United States)

    Varela, Juan A; Ferreira, Joana S; Dupuis, Julien P; Durand, Pauline; Bouchet, Delphine; Groc, Laurent

    2016-10-01

    Recent developments in single-molecule imaging have revealed many biological mechanisms, providing high spatial and temporal resolution maps of molecular events. In neurobiology, these techniques unveiled that plasma membrane neurotransmitter receptors and transporters laterally diffuse at the surface of cultured brain cells. The photostability of bright nanoprobes, such as quantum dots (QDs), has given access to neurotransmitter receptor tracking over long periods of time with a high spatial resolution. However, our knowledge has been restricted to cultured systems, i.e., neurons and organotypic slices, therefore lacking several aspects of the intact brain rheology and connectivity. Here, we used QDs to track single glutamatergic [Formula: see text]-methyl-d-aspartate receptors (NMDAR) in acute brain slices. By delivering functionalized nanoparticles in vivo through intraventricular injections to rats expressing genetically engineered-tagged NMDAR, we successfully tracked the receptors in native brain tissue. Comparing NMDAR tracking to different classical brain preparations (acute brain slices, cultured organotypic brain slices, and cultured neurons) revealed that the surface diffusion properties shared several features and are also influenced by the nature of the extracellular environment. Together, we describe the experimental procedures to track plasma membrane NMDAR in dissociated and native brain tissue, paving the way for investigations aiming at characterizing receptor diffusion biophysics in intact tissue and exploring the physiopathological roles of receptor surface dynamics. PMID:27429996

  3. Study of Triclabendazole (TCBZ Effect on Aspartate Transaminase (AST Activity of Fasciola gigantica Parasite and Liver Enzyme Activity Assay

    Directory of Open Access Journals (Sweden)

    Shima Shafaei

    2015-10-01

    Full Text Available  Background: Aspartate transaminase (AST is an important enzyme in parasite and liver tissue. The purpose of this investigation is to evaluate triclabendazole (TCBZ effect on AST activity of Fasciola gigantica parasite. To compare of enzyme level of parasite and its host tissue, enzyme activity of F. gigantica parasite and liver tissues were also determined. Method:The livers were collected from sheep slaughtered in local slaughterhouse and living F. gigantica parasites were isolated. The washed parasites were cultured in buffe rmedia with or without Triclabendazole (Egaten®; 15μg/mL in an incubator at 37° C. Extractions of collected parasites and liver tissues were prepared by homogenizing buffer in a Mortar and pestle. Extraction samples were examined for protein measurement, AST activity assay and protein recognition. Results:The results of AST assay revealed, enzyme activity for treated and untreated is not significant. Healthy liver tissue shows significantly higher enzyme activity than parasite. Enzyme activity for healthy and infected liver tissues was significant. Enzymatic proteins including Cathepsin L & B (Protease were recognized in parasite samples. Conclusion:Although AST could not be concerned as an indicator for efficiency treatment, however may be involved as a biomarker for biochemical comparison of parasite and host tissue.

  4. Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

    Science.gov (United States)

    Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

    2013-02-01

    Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature. PMID:22648335

  5. A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression in Pichia pastoris.

    Science.gov (United States)

    Yegin, Sirma; Fernandez-Lahore, Marcelo

    2013-06-01

    In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.

  6. Development of an Electrochemical-Based Aspartate Aminotransferase Nanoparticle Ir-C Biosensor for Screening of Liver Diseases

    Directory of Open Access Journals (Sweden)

    Chung-Chiun Liu

    2012-05-01

    Full Text Available Aspartate aminotransaminase (AST is a hepatocelluar enzyme released into the bloodstream when hepatic cells are damaged, resulting in elevated blood levels of AST. A single use, disposable biosensor prototype, composed of catalytic iridium nano-particles dispersed on carbon paste, was developed to detect enzymatically-produced H2O2 in AST-mediated reactions. This biosensor is capable of measuring AST levels in a phosphate buffer and undiluted human serum over the concentration range of 0 to 0.89 μg/mL AST concentration (corresponding to 0–250 UL−1 specific activity. The biosensor operates at relatively low oxidation potential (+0.3 volt (V versus the printed Ag/AgCl, minimizing any potential chemical interference in human serum. The measurements of AST in human serum using the biosensor compared well with those measured by standard hospital spectrophotometric assays. This Ir-C biosensor may be useful for AST measurements in the clinical environment.

  7. Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro

    Institute of Scientific and Technical Information of China (English)

    Jie-li LI; Lin ZHAO; Bin CUI; Lian-fu DENG; Guang NING; Jian-min LIU

    2011-01-01

    Aim: Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro.Methods: Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression.The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit.Results: Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100μmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 μmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 μmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L).Conclusion: The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.

  8. Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

    Science.gov (United States)

    Babaei, H; Mansouri-Najand, L; Molaei, M M; Kheradmand, A; Sharifan, M

    2007-05-01

    This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis. PMID:17268916

  9. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    Science.gov (United States)

    Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

    2016-01-01

    Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

  10. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1.

    Directory of Open Access Journals (Sweden)

    Fengjuan Zhang

    2016-01-01

    Full Text Available Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins are essential components of Bacillus thuringiensis (Bt biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1. In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

  11. Marked diversity in the action of growth factors on N-methyl-D-aspartate-induced neuronal degeneration.

    Science.gov (United States)

    Prehn, J H

    1996-06-13

    Neuronal degeneration was induced in cultured rat hippocampal neurons by a 20-min exposure to the glutamatergic agonist, N-methyl-D-aspartate (NMDA; 100 microM), and the neuroprotective activity of a set growth factors and cytokines was compared. During the early stages of degeneration, NMDA induced changes that were characteristic of neuronal necrosis, including swelling and darkening of the neuronal soma and swelling of neurites, leading to the formation of beaded varicosities ('blebs'). These changes were followed by nuclear pyknosis, formation of double-stranded DNA breaks and loss of membrane integrity. Only transforming growth factor-beta 1 (TGF-beta 1; 1-10 ng/ml) and tumor necrosis factor-alpha (TNF-alpha; 30 ng/ml) protected the hippocampal neurons against NMDA neurotoxicity after short-term (60 min) pre-treatments. Interleukin-1 beta (10-100 ng/ml) and fibroblast growth factor-2 (FGF-2; 50 ng/ml) were clearly effective when administered 24 h prior to the NMDA exposure, but not when given 60 min before the insult. Interestingly, the protective effect of interleukin-1 beta was significantly reduced in the presence of a neutralizing antibody to TGF-beta. Of note, short-term pre-treatment with brain-derived neurotrophic factor (BDNF; 5-50 ng/ml) significantly potentiated NMDA-induced neurodegeneration. These experiments demonstrate marked diversity in the actions of growth factors on NMDA-induced neuronal degeneration. PMID:8813618

  12. Aspartate aminotransferase-to-platelet ratio index for fibrosis and cirrhosis prediction in chronic hepatitis C patients

    Directory of Open Access Journals (Sweden)

    Roberto Gomes da Silva Junior

    2008-02-01

    Full Text Available In chronic hepatitis C (CHC, liver biopsy is the gold standard method for assessing liver histology, however it is invasive and can have complications. Non-invasive markers have been proposed and aspartate aminotransferase (AST-to-platelet ratio index (APRI has been shown as an easy and inexpensive marker of liver fibrosis. This study evaluated the diagnostic performance of APRI for significant fibrosis and cirrhosis prediction in CHC patients. This study included treatment-naive CHC patients who had undergone liver biopsy from January 2000 to August 2006. All histological slides were reviewed according to the METAVIR system. APRI was calculated based on laboratory results performed within four months from the biopsy. Twenty-eight (56% patients had significant fibrosis (F2-F4 and 13 (26% had cirrhosis (F4. The area under ROC curves of APRI for predicting significant fibrosis and cirrhosis were 0.92 (0.83-1.00 and 0.92 (0.85-1.00, respectively. Using cut-off values recommended by prior studies, significant fibrosis could be identified, in accordance with liver biopsy, in 44% and cirrhosis in 66% of patients. APRI could identify significant fibrosis and cirrhosis at a high degree of accuracy in studied patients.

  13. Voltammetric determination of dopamine and norepinphrine on a glassy carbon electrode modified with poly (L-aspartic acid)

    Indian Academy of Sciences (India)

    Zhangyu Yu; Xiaochun Li; Xueliang Wang; Xinying Ma; Xia Li; Kewei Cao

    2012-03-01

    A convenient and useful method for the voltammetric determination of dopamine (DA) and norepinphrine (NE) based on poly(L-aspartic acid) modified glassy carbon electrode (GCE) is reported in this paper. The modified electrode exhibits excellent electro-catalytic activities for the oxidation-reduction of DA and NE, as well as eliminating the interference of ascorbic acid (AA) and uric acid (UA). Factors influencing the detection processes are optimized and the kinetic parameters are calculated. Under the optimal conditions, the anodic peak currents of DA and NE are linear with their concentration and the detection limits (S/N = 3) are 1.0 × 10−9 mol L-1 for DA and 4.31 10−9 mol L-1 for NE, respectively. The practical application of this method is demonstrated by determining the concentration of NE and DA in injection which is commercially available with satisfactory results. Compared with other electrochemical methods, this method is simple, highly selective and sensitive.

  14. N-methyl-D-aspartate receptor subtype 3A promotes apoptosis in developing mouse brain exposed to hyperoxia

    Institute of Scientific and Technical Information of China (English)

    Jimei Li; Shanping Yu; Zhongyang Lu; Osama Mohamad; Ling Wei

    2012-01-01

    In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyl-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling (TUNEL)/neuron-specific nuclear protein (NeuN) and 5-bromo-2'-deoxyuridine (BrdU)/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL+/NeuN+ and BrdU+/NeuN+ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.

  15. Src, a Molecular Switch Governing Gain Control of Synaptic Transmission Mediated by N-methyl-D-Aspartate Receptors

    Science.gov (United States)

    Yu, Xian-Min; Salter, Michael W.

    1999-07-01

    The N-methyl-D-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

  16. Modulation of NR1 Subunit of N-Methyl-D-Aspartate Receptor by Ovariectomy and Passive Avoidance Learning

    Directory of Open Access Journals (Sweden)

    Mahnaz Taherianfard

    2012-10-01

    Full Text Available BACKGROUND: Learning and memory are the most intensively studied subjects in neuroscience. Various approaches have been used to understand the underlying cellular and molecular mechanisms. Numerous studies have shown that different sites of brain are involved in learning and memory mechanisms. Two sites of mammalian brain that show high density of these receptors are CA1 region of hippocampus and Purkinje cell layer of cerebellum.METHODS: Twenty four Sprague-Dawley rats were used in 4 groups: Control-1 (intact without learning; control-2 (intact with learning; ovariectomy (OVX without learning and OVX with learning. A shuttle box apparatus used for passive avoidance learning procedure. Immunohistochemical procedure was used for determination of NR1 subunit of N-methyl-D-aspartate (NMDA receptor. Photoshop software was used for determination of color intensity.RESULTS: Immunohistological finding of this experiment indicated that OVX has a negative effect on density of NR1 subunit of NMDA receptors in two brain regions. Other finding of this study showed that passive avoidance learning significantly increased density of NR1 subunit of NMDA receptors in two brain regions.CONCLUSION: These results indicated that the sex hormone can modulate function and expression of the NR1 subunit of NMDA receptor in CA1 region of hippocampus and Purkinje cell layer of cerebellum.

  17. Quantum Computational Calculations of the Ionization Energies of Acidic and Basic Amino Acids: Aspartate, Glutamate, Arginine, Lysine, and Histidine

    Science.gov (United States)

    de Guzman, C. P.; Andrianarijaona, M.; Lee, Y. S.; Andrianarijaona, V.

    An extensive knowledge of the ionization energies of amino acids can provide vital information on protein sequencing, structure, and function. Acidic and basic amino acids are unique because they have three ionizable groups: the C-terminus, the N-terminus, and the side chain. The effects of multiple ionizable groups can be seen in how Aspartate's ionizable side chain heavily influences its preferred conformation (J Phys Chem A. 2011 April 7; 115(13): 2900-2912). Theoretical and experimental data on the ionization energies of many of these molecules is sparse. Considering each atom of the amino acid as a potential departing site for the electron gives insight on how the three ionizable groups affect the ionization process of the molecule and the dynamic coupling between the vibrational modes. In the following study, we optimized the structure of each acidic and basic amino acid then exported the three dimensional coordinates of the amino acids. We used ORCA to calculate single point energies for a region near the optimized coordinates and systematically went through the x, y, and z coordinates of each atom in the neutral and ionized forms of the amino acid. With the calculations, we were able to graph energy potential curves to better understand the quantum dynamic properties of the amino acids. The authors thank Pacific Union College Student Association for providing funds.

  18. Synthesis and characterization of magnetic and luminescent Fe3O4/CdTe nanocomposites using aspartic acid as linker

    Institute of Scientific and Technical Information of China (English)

    Xiu Ling Wang; Lu Wei; Guan Hong Tao; Meng Qiong Huang

    2011-01-01

    In this study, the preparation of a new kind of magnetic and luminescent Fe3O4/CdTe nanocomposites was demonstrated. Superparamagnetic Fe3O4 nanoparticles were first synthesized by hydrothermal coprecipitation of ferric and ferrous ions, followed by the modification of their surfaces with tetramethylammonium hydroxide (TMAOH) and the chemical activation with aspartic acid. The surface-modified Fe3O4 nanoparticles were then covalently coated with CdTe quantum dots (QDs), which were modified with mercaptoacetic acid (MPA), to form the Fe3O4/CdTe magnetic and luminescent nanocomposites through the coordination of the amino groups on the surfaces of Fe3O4 and the carboxyl groups on CdTe QDs. The structure and properties of as-synthesized nanocomposites were characterized. It was indicated that the nanocomposites possessed structure with an average diameter of 40-50 nm, yellow-green emission feature and room temperature ferro-magnetism. Both the fluorescence and UV-vis absorption spectra of the nanocomposites showed a blue shift comparing with those of CdTe QDs. The mechanism of the blue shift was presented. The nanocomposites retained the ferromagnetic property with a saturation magnetization of 8.9 emu/g.

  19. Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

    Science.gov (United States)

    Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-Ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

    2016-04-01

    Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians. PMID:26711235

  20. Initiating or Switching to Biphasic Insulin Aspart 30/70 Therapy in Subjects with Type 2 Diabetes Mellitus. An Observational Study

    DEFF Research Database (Denmark)

    Breum, Leif; Almdal, Thomas; Eiken, Pia;

    2008-01-01

    OBJECTIVE: To investigate tolerability and glycemic control over 26 weeks in patients with type 2 diabetes (T2D) who initiated insulin with, or switched to, biphasic insulin aspart 30/70 (BIAsp 30) in routine clinical care. METHODS: This was a non-randomized, non-interventional, open-label, obser......OBJECTIVE: To investigate tolerability and glycemic control over 26 weeks in patients with type 2 diabetes (T2D) who initiated insulin with, or switched to, biphasic insulin aspart 30/70 (BIAsp 30) in routine clinical care. METHODS: This was a non-randomized, non-interventional, open...... adverse drug reactions (SADR), glycemic parameters and hypoglycemic events were obtained from patients' notes, patients' diaries and recall, and transferred to case report forms by physicians at baseline (during 4 weeks prior to BIAsp 30 therapy) and after 12 and 26 weeks of treatment. RESULTS: 421...

  1. [Effect of excitant amino acid antagonists on glutamate receptors in the locust and on convulsions induced by glutamate, aspartate, kynurenine and quinolinic acid in mice].

    Science.gov (United States)

    Ryzhov, I V; Slepokurov, M V; Lapin, I P; Mandel'shtam, Iu E; Aleksandrov, V G

    1986-03-01

    All excitatory amino acid antagonists studied: diethyl esters of aspartic (DEEA) and glutamic (DEEG) acids, 2-amino-3-phosphono-propionic acid (APPA) and 2-amino-4-phosphono-butanoic acid (APBA), diminished the amplitude of excitatory postsynaptic potentials (EPP) of the locust (Locusta migratoria migratorioides) muscle fibers and arbitrary blocked glutamate (GLU) and aspartate (ASP) responses. Kynurenine (KYN) and quinolinic (QUI) acid had no effect on EPP even at a concentration of 2 X 10(-2) M. The antagonists were not strictly selective against intracerebroventricularly administered endogenous convulsants: GLU, ASP, KYN and QUI and in simulation of experimental seizures in mice. The antagonists structurally similar to ASP prevented ASP- and KYN-induced seizures in lower doses than GLU derivatives. Anti-KYN, but not anti-QUI DEEA, DEEG, APPA and APBA efficacy suggests that KYN and QUI act on different structures or binding sites. PMID:2869799

  2. Isolation and structural analysis of a gene coding for a novel type of aspartic proteinase from buckwheat seed (Fagopyrum esculentum Moench

    Directory of Open Access Journals (Sweden)

    Milisavljević Mira Đ.

    2007-01-01

    Full Text Available A novel type of aspartic proteinase gene was isolated from the cDNA library of developing buckwheat seeds. This cDNA, FeAPL1, encoded an AP-like protein lacking the plant-specific insert (PSI domain characteristic of typical plant aspartic proteinases. In addition the corresponding genomic fragment was isolated. It is demonstrated that this gene does not contain introns. Since bioinformatics analysis of the Arabidopsis genome showed that most potential AP genes are intronless and PSI-less, it appears that "atypical" is an inappropriate word for that class of AP. Isolation of this specific buckwheat gene among the small group of those isolated from other plant species provides a new perspective on the diversity of AP family members in plants. .

  3. Inhibition mechanism analysis & research of the poly aspartic acid corrosion inhibitor%聚天冬氨酸缓蚀剂缓蚀机理分析研究

    Institute of Scientific and Technical Information of China (English)

    王娴

    2012-01-01

    文中介绍了聚天冬氨酸缓蚀剂缓蚀机理,并对聚天冬氨酸缓蚀剂机理的研究现状以及发展趋势进行了综述。%It was introduced inhibition mechanism of the poly aspartic acid corrosion inhibitor. Mean- while, it was summarizeed the research progress of the inhibition meehanismf the poly aspartic acid cor- rosion inhibitor and the development trend of the poly aspartic acid corrosion inhibitor in this article.

  4. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis)

    OpenAIRE

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-01-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis ...

  5. Dihydroorotase from the Hyperthermophile Aquifiex aeolicus Is Activated by Stoichiometric Association with Aspartate Transcarbamoylase and Forms a One-Pot Reactor for Pyrimidine Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengfei; Martin, Philip D.; Purcarea, Cristina; Vaishnav, Asmita; Brunzelle, Joseph S.; Fernando, Roshini; Guy-Evans, Hedeel I.; Evans, David R.; Edwards, Brian F.P.; (WSU-MED); (IB-Bucharest); (NWU); (E.Mich.U.)

    2009-08-14

    In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 {angstrom} resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 {angstrom} in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

  6. L-Aspartic and l-glutamic acid ester-based ProTides of anticancer nucleosides: Synthesis and antitumoral evaluation.

    Science.gov (United States)

    Gao, Ling-Jie; De Jonghe, Steven; Daelemans, Dirk; Herdewijn, Piet

    2016-05-01

    A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity. PMID:27032331

  7. Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population

    OpenAIRE

    Yongfeng Yang; Wenqiang Li; Hongxing Zhang; Ge Yang; Xiujuan Wang; Minli Ding; Tianzi Jiang; Luxian Lv

    2015-01-01

    Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In ...

  8. Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

    2007-01-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

  9. Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

    Energy Technology Data Exchange (ETDEWEB)

    Julin, D.A.; Wiesinger, H.; Toney, M.D.; Kirsch, J.F. (Univ. of California, Berkeley (USA))

    1989-05-02

    The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.

  10. Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones.

    OpenAIRE

    Orser, B. A.; Bertlik, M.; Wang, L. Y.; MacDonald, J. F.

    1995-01-01

    1. The effects of propofol (2,6 di-isopropylphenol) on responses to the selective glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, were investigated in cultured hippocampal neurones of the mouse. Whole cell and single channel currents were recorded by patch-clamp techniques. Drugs were applied with a multi-barrel perfusion system. 2. Propofol produced a reversible, dose-dependent inhibition of whole cell currents activated by NMDA. The concentration of propofol which indu...

  11. Complementary roles of neurotrophin 3 and a N-methyl-d-aspartate antagonist in the protection of noise and aminoglycoside-induced ototoxicity

    OpenAIRE

    Duan, Maoli; Agerman, Karin; Ernfors, Patrik; Canlon, Barbara

    2000-01-01

    Recent progress has been made regarding the prevention of hearing loss. However, the complete protection of both hair cells and spiral ganglion neurons, with restored function, has not yet been achieved. It has been shown that spiral ganglion neuronal loss can be prevented by neurotrophin 3 (NT3) and hair cell damage by N-methyl-d-aspartate (NMDA) receptor antagonists. Here we demonstrate that the combined treatment with MK801, a NMDA antagonist, and NT3 protect both cochlear morphology and p...

  12. Regulatory structure of the biosynthetic pathway for the aspartate family of amino acids in Lemna paucicostata Hegelm. 6746, with special reference to the role of aspartokinase

    International Nuclear Information System (INIS)

    Comprehensive studies were made with Lemna paucicostate Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme along being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [14C]threonine and [14C]homoserine in Lemna

  13. The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

    Science.gov (United States)

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-11-15

    Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme. PMID:27283639

  14. Intersubunit communication in the dihydroorotase-aspartate transcarbamoylase complex of Aquifex aeolicus: Intersubunit Communication in a Pyrimidine Biosynthetic Complex

    Energy Technology Data Exchange (ETDEWEB)

    Evans, Hedeel Guy [Department of Chemistry, Eastern Michigan University, Ypsilanti Michigan 48197; Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit Michigan 48201; Fernando, Roshini [Department of Chemistry, Eastern Michigan University, Ypsilanti Michigan 48197; Vaishnav, Asmita [Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit Michigan 48201; Kotichukkala, Mahalakshmi [Department of Chemistry, Eastern Michigan University, Ypsilanti Michigan 48197; Heyl, Deborah [Department of Chemistry, Eastern Michigan University, Ypsilanti Michigan 48197; Martin, Philip D. [Department of Chemistry, Wayne State University, Detroit Michigan 48202; Hachem, Fatme [Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit Michigan 48201; Brunzelle, Joseph S. [Life Sciences Collaborative Access Team, Northwestern University, Center for Synchrotron Research, Argonne Illinois 60439; Edwards, Brian F. P. [Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit Michigan 48201; Evans, David R. [Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit Michigan 48201

    2013-12-19

    Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (Ki = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.

  15. Ligand-functionalized degradable polyplexes formed by cationic poly(aspartic acid)-grafted chitosan-cyclodextrin conjugates

    Science.gov (United States)

    Song, Hai-Qing; Li, Rui-Quan; Duan, Shun; Yu, Bingran; Zhao, Hong; Chen, Da-Fu; Xu, Fu-Jian

    2015-03-01

    Polypeptide-based degradable polyplexes attracted considerable attention in drug delivery systems. Polysaccharides including cyclodextrin (CD), dextran, and chitosan (CS) were readily grafted with cationic poly(aspartic acid)s (PAsps). To further enhance the transfection performances of PAsp-based polyplexes, herein, different types of ligand (folic acid, FA)-functionalized degradable polyplexes were proposed based on the PAsp-grafted chitosan-cyclodextrin conjugate (CCPE), where multiple β-CDs were tied on a CS chain. The FA-functionalized CCPE (i.e., CCPE-FA) was obtained via a host-guest interaction between the CD units of CCPE and the adamantane (Ad) species of Ad-modified FA (Ad-FA). The resulting CCPE/pDNA, CCPE-FA/pDNA, and ternary CCPE-FA/CCPE/pDNA (prepared by layer-by-layer assembly) polyplexes were investigated in detail using different cell lines. The CCPE-based polyplexes displayed much higher transfection efficiencies than the CS-based polyplexes reported earlier by us. The ternary polyplexes of CCPE-FA/CCPE/pDNA produced excellent gene transfection abilities in the folate receptor (FR)-positive tumor cells. This work would provide a promising means to produce highly efficient polyplexes for future gene therapy applications.Polypeptide-based degradable polyplexes attracted considerable attention in drug delivery systems. Polysaccharides including cyclodextrin (CD), dextran, and chitosan (CS) were readily grafted with cationic poly(aspartic acid)s (PAsps). To further enhance the transfection performances of PAsp-based polyplexes, herein, different types of ligand (folic acid, FA)-functionalized degradable polyplexes were proposed based on the PAsp-grafted chitosan-cyclodextrin conjugate (CCPE), where multiple β-CDs were tied on a CS chain. The FA-functionalized CCPE (i.e., CCPE-FA) was obtained via a host-guest interaction between the CD units of CCPE and the adamantane (Ad) species of Ad-modified FA (Ad-FA). The resulting CCPE/pDNA, CCPE

  16. Tumor progression-related transmembrane protein aspartate β-hydroxylase is a target for immunotherapy of hepatocellular carcinoma

    Science.gov (United States)

    Shimoda, Masafumi; Tomimaru, Yoshito; Charpentier, Kevin P.; Safran, Howard; Carlson, Rolf I.; Wands, Jack

    2012-01-01

    Background/Aims Hepatocellular carcinoma (HCC) has a poor survival rate due to recurrent intrahepatic metastases and lack of effective adjuvant therapy. Aspartate-β-hydroxylase (ASPH) is an attractive cellular target since it is a highly conserved transmembrane protein overexpressed on both murine and human HCC tumors, and promotes a malignant phenotype as characterized by enhanced tumor cell migration and invasion. Methods Dendritic cells (DCs), expanded and isolated from the spleen, were incubated with a cytokine cocktail to optimize IL-12 secretion and co-stimulatory molecule expression, then subsequently loaded with ASPH protein for immunization. Mice were injected with syngeneic BNL HCC tumor cells followed by subcutaneous inoculation with 5–10×105 ASPH loaded DCs using a prophylactic and therapeutic experimental approach. Tumor infiltrating lymphocytes (TILs) were characterized, and their role in producing anti-tumor effects determined. The immunogenicity of ASPH protein with respect to activating antigen specific CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) was also explored. Methods We found that immunotherapy with ASPH-loaded DCs suppressed and delayed established HCC and tumor growth when administered prophylactically. Ex-vivo re-stimulation experiments and in vivo depletion studies demonstrate that both CD4+ and CD8+ cells contributed to anti-tumor effects. Using PBMCs derived from healthy volunteers and HCC patients, we showed that ASPH stimulation led to significant development of antigen-specific CD4+ T-cells. Conclusion Immunization with ASPH-loaded DCs has substantial anti-tumor effects which could reduce the risk of HCC recurrence. PMID:22245894

  17. Computational study on 3D structure of L-aspartic acid and L-glutamic acid: molecular descriptors and properties

    Directory of Open Access Journals (Sweden)

    Stefaniu Amalia

    2016-06-01

    Full Text Available The aim of this work is to provide a comprehensive and complex analysis of molecular descriptors and properties of two similar amino acids, L-Aspartic acid and L-Glutamic acid, using a software tool for calculations and properties predictions. As amino acids are model compounds for predicting the physical-chemical properties and behavior of biological, larger molecules as peptides or proteins, researches were focused on providing accurate mechanical calculations using: molecular/mechanical methods. Our study aims to initiate a linear scaling approach, by dividing a large system into small subsystems and performing the calculations for each, individually, then, embedding and correcting the information globally. The calculations were performed on the 3D structure of the studied amino acids that were first generated, as CPK model, and optimized by energy minimization. A comparative assay on their topological, molecular descriptors and properties was conducted, in vacuum and in water, using the Hartree-Fock model and second-order Møller-Plesset perturbation theory MP2 for predicting structure, energy and property calculations with Spartan’14 software. Values of molecular properties such as area, volume, polar surface area, polarizability, ovality, logP, dipole moment, HOMO-LUMO gap, distances and angles between atoms, were obtained. The results have been interpreted in terms of electronic effects of side chain groups, molecular deformability, steric factors and reactivity. This approach can be extended to other amino acids in order to predict protein-ligand interactions, important aspects in drug design studies and protein engineering.

  18. [Clinical course of recovery from cognitive dysfunction in a patient with anti-N-methyl-D-aspartate receptor encephalitis].

    Science.gov (United States)

    Asai, Chikako; Morinaga, Akiyoshi; Yamamoto, Kumiko; Imamura, Toru

    2014-10-01

    Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is an autoimmune disorder, which occurs commonly in young women and is often associated with ovarian teratomas. We report the case of a patient with this disease, who exhibited cognitive deficits, and describe the clinical course of recovery from cognitive dysfunction. A 29-year-old right-handed woman suffered from chills and fever for 7 days prior to admission to hospital, and complained that she could not understand the content of TV programs. Following admission to hospital, she was found to have an ovarian teratoma and underwent oophorectomy. She was diagnosed with anti-NMDA receptor encephalitis based on the presence of antibodies in the serum and cerebrospinal fluid. She subsequently experienced phases with disturbance of consciousness and involuntary movement, and then moved into the gradual recovery phase 3 months after onset. Cerebral SPECT revealed a left-dominant decrease of blood flow in the prefrontal regions bilaterally. Neuropsychological examination 3 months after onset revealed frontal lobe syndrome comprising executive dysfunction, decreased spontaneity, and environmental dependency in addition to recent memory deficits. Approximately 6 months after onset, recent memory impairments and environmental dependency were resolved, and a gradual improvement in spontaneity and executive function was seen. One year after onset, the patient had regained independence and ability to self-care, and returned to her workplace. Our observations suggest that patients with anti-NMDA receptor encephalitis may recover from frontal lobe syndrome, including executive dysfunction and decreased spontaneity, slower than patients with other cognitive dysfunctions do. PMID:25296876

  19. Antagonist properties of Conus parius peptides on N-methyl-D-aspartate receptors and their effects on CREB signaling.

    Directory of Open Access Journals (Sweden)

    Shailaja Kunda

    Full Text Available Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR. We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg(2+. Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg(2+. Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca(2+ (iCa(2+ influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A(-/- and GluN2B(-/- mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A(-/--derived mouse neurons, as compared to those isolated from GluN2B(-/--mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser(133, suggesting that the peptides modulated iCa(2+ entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa(2+ influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.

  20. Sequence diversities of serine-aspartate repeat genes among Staphylococcus aureus isolates from different hosts presumably by horizontal gene transfer.

    Directory of Open Access Journals (Sweden)

    Huping Xue

    Full Text Available BACKGROUND: Horizontal gene transfer (HGT is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr family has been compared among different sources of Staphylococcus aureus (S. aureus to discover sequence diversities within their genomes. METHODOLOGY/PRINCIPAL FINDINGS: Four sdr genes were analyzed for 21 different S. aureus strains and 218 mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122 and mastitis isolates in this study, ovine mastitis (ED133, pig (ST398, chicken (ED98, and human methicillin-resistant S. aureus (MRSA (TCH130, MRSA252, Mu3, Mu50, N315, 04-02981, JH1 and JH9 were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. CONCLUSIONS: Certain sdr gene sequences were shared in S. aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may

  1. The efficacy of aspartate aminotransferase-toplatelet ratio index for assessing hepatic fibrosis in childhood nonalcoholic steatohepatitis for medical practice

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    Earl Kim

    2013-01-01

    Full Text Available Purpose: Childhood obesity is associated with nonalcoholic fatty liver disease (NAFLD, and it has become one of the most common causes of childhood chronic liver diseases which significant as a cause of liver related mortality and morbidity in children in the United States. The development of simpler and easier clinical indices for medical practice is needed to identify advanced hepatic fibrosis in childhood NAFLD instead of invasive method like liver biopsy. FibroScan and aspartate aminotransferase (AST-to-platelet ratio index (APRI have been proposed as a simple and noninvasive predictor to evaluate hepatic fibrosis in several liver diseases. APRI could be a good alternative to detect pathologic change in childhood NAFLD. The purpose of this study is to validate the efficacy of APRI for assessing hepatic fibrosis in childhood NAFLD based on FibroScan. Methods: This study included 23 children with NAFLD who underwent FibroScan. Clinical, laboratory and radiological evaluation including APRI was performed. To confirm the result of this study, 6 patients received liver biopsy. Results: Factors associated with hepatic fibrosis (stiffness measurement &gt;5.9 kPa Fibroscan were triglyceride, AST, alanine aminotransferase, platelet count, APRI and collagen IV. In multivariate analysis, APRI were correlated with hepatic fibrosis (&gt;5.9 kPa. In receiver operating characteristics curve, APRI of meaningful fibrosis (cutoff value, 0.4669; area under the receiver operating characteristics, 0.875 presented sensitivity of 94%, specificity of 66%, positive predictive value of 94%, and negative predictive value of 64%. Conclusion: APRI might be a noninvasive, simple, and readily available method for medical practice to predict hepatic fibrosis of childhood NAFLD.

  2. Glutamate-N-methyl-D-aspartate receptor modulation and minocycline for the treatment of patients with schizophrenia: an update

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    C. Chaves

    2009-11-01

    Full Text Available Growing consistent evidence indicates that hypofunction of N-methyl-D-aspartate (NMDA transmission plays a pivotal role in the neuropathophysiology of schizophrenia. Hence, drugs which modulate NMDA neurotransmission are promising approaches to the treatment of schizophrenia. The aim of this article is to review clinical trials with novel compounds acting on the NMDA receptor (NMDA-R. This review also includes a discussion and translation of neuroscience into schizophrenia therapeutics. Although the precise mechanism of action of minocycline in the brain remains unclear, there is evidence that it blocks the neurotoxicity of NMDA antagonists and may exert a differential effect on NMDA signaling pathways. We, therefore, hypothesize that the effects of minocycline on the brain may be partially modulated by the NMDA-R or related mechanisms. Thus, we have included a review of minocycline neuroscience. The search was performed in the PubMed, Web of Science, SciELO, and Lilacs databases. The results of glycine and D-cycloserine trials were conflicting regarding effectiveness on the negative and cognitive symptoms of schizophrenia. D-serine and D-alanine showed a potential effect on negative symptoms and on cognitive deficits. Sarcosine data indicated a considerable improvement as adjunctive therapy. Finally, minocycline add-on treatment appears to be effective on a broad range of psychopathology in patients with schizophrenia. The differential modulation of NMDA-R neurosystems, in particular synaptic versus extrasynaptic NMDA-R activation and specific subtypes of NMDA-R, may be the key mediators of neurogenesis and neuroprotection. Thus, psychotropics modulating NMDA-R neurotransmission may represent future monotherapy or add-on treatment strategies in the treatment of schizophrenia.

  3. Adenosine A1 receptor activation modulates N-methyl-d-aspartate (NMDA) preconditioning phenotype in the brain.

    Science.gov (United States)

    Constantino, Leandra C; Pamplona, Fabrício A; Matheus, Filipe C; Ludka, Fabiana K; Gomez-Soler, Maricel; Ciruela, Francisco; Boeck, Carina R; Prediger, Rui D; Tasca, Carla I

    2015-04-01

    N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.

  4. Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor.

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    Trammell, M A; Falke, J J

    1999-01-01

    Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU

  5. N-methyl-D-aspartate increases acetylcholine release from rat striatum and cortex: its effect is augmented by choline

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    Ulus, I. H.; Buyukuysal, R. L.; Wurtman, R. J.

    1992-01-01

    We examined the effects of N-methyl-D-aspartate (NMDA), a glutamate agonist, and of glutamate itself, on acetylcholine (ACh) release from superfused rat striatal slices. In a Mg(++)-free medium, NMDA (32-1000 microM) as well as glutamate (1 mM) increased basal ACh release by 35 to 100% (all indicated differences, P less than .05), without altering tissue ACh or choline contents. This augmentation was blocked by Mg++ (1.2 mM) or by MK-801 (10 microM). Electrical stimulation (15 Hz, 75 mA) increased ACh release 9-fold (from 400 to 3660 pmol/mg of protein): this was enhanced (to 4850 pmol/mg of protein) by NMDA (100 microM). ACh levels in stimulated slices fell by 50 or 65% depending on the absence or presence of NMDA. The addition of choline (40 microM) increased ACh release both basally (570 pmol/mg of protein) and with electrical stimulation (6900 pmol/mg of protein). In stimulated slices choline acted synergistically with NMDA, raising ACh release to 10,520 pmol/mg of protein. The presence of choline also blocked the fall in tissue ACh. No treatment affected tissue phospholipid or protein levels. NMDA (32-320 microM) also augmented basal ACh release from cortical but not hippocampal slices. Choline efflux from striatal and cortical (but not hippocampal) slices decreased by 34 to 50% in Mg(++)-free medium. These data indicate that NMDA-like drugs may be useful, particularly in combination with choline, to enhance striatal and cortical cholinergic activity. ACh release from rat hippocampus apparently is not affected by NMDA receptors.

  6. Effect of rifaximin, probiotics, and l-ornithine l-aspartate on minimal hepatic encephalopathy: A randomized controlled trial

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    Kapil Sharma

    2014-01-01

    Full Text Available Background/Aims: Minimal hepatic encephalopathy (MHE implies subtle impairment of cognitive functions in the absence of features of overt encephalopathy. We aimed to determine the prevalence of MHE in patients with liver cirrhosis and to find out the effect of rifaximin, probiotics, and l-ornithine l-aspartate (LOLA individually in reversal of MHE by comparing it with placebo group. Patients and Methods: This study was carried out in two phases. Phase I included the recruitment of 250 apparently healthy controls and extraction of normative data utilizing three neuropsychometric tests (NPTs and critical flicker frequency (CFF test. Phase II consisted of screening and recruitment of patients of MHE followed by drugs trial. A total of 317 cirrhotics were screened; 111 were excluded and the remaining 206 cirrhotics were screened for MHE using NPTs and/or CFF test. Of these, 124 patients with MHE were randomized to receive LOLA (n = 31, rifaximin (n = 31, probiotics (n = 32, for 2 months and were compared with patients who were given placebo (n = 30. Results: Out of 206 cirrhotics, 124 (60.19% had MHE. Among these 124 MHE patients, 87 (70.16% patients had CFF <39Hz, 112 (90.32% patients with MHE had two or more abnormal NPTs, and 75 (60.48% patients had abnormality on both the CFF values and more than two abnormal NPTs. Intention-to-treat analysis showed the number of patients who improved after giving treatment were 67.7% (21/31, 70.9% (22/31, 50% (16/32, and 30% (9/30 for LOLA, rifaximin, probiotics, and placebo, respectively. CFF scores and improvement in psychometric tests after treatment were significantly higher (P < 0.05 for LOLA, rifaximin, and probiotics as compared with placebo group. Conclusions: Prevalence of MHE is high in patients with cirrhosis of liver. Rifaximin, LOLA, and probiotics are better than giving placebo in patients with MHE.

  7. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine.

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    Savignac, Helene M; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P E; Tzortzis, George; Burnet, Philip W J

    2013-12-01

    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and

  8. Mercury-induced toxicity of rat cortical neurons is mediated through N-methyl-D-Aspartate receptors

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    Xu Fenglian

    2012-09-01

    Full Text Available Abstract Background Mercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer’s disease, Parkinson’s disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid on cultured, rat cortical neurons. Results We found that inorganic mercuric chloride (HgCl2 –at 0.025 to 25 μM not only caused neuronal degeneration but also perturbed neuronal excitability. Whole-cell patch-clamp recordings of pyramidal neurons revealed that HgCl2 not only enhanced the amplitude and frequency of synaptic, inward currents, but also increased spontaneous synaptic potentials followed by sustained membrane depolarization. HgCl2 also triggered sustained, 2–5 fold rises in intracellular calcium concentration ([Ca2+]i. The observed increases in neuronal activity and [Ca2+]i were substantially reduced by the application of MK 801, a non-competitive antagonist of N-Methyl-D-Aspartate (NMDA receptors. Importantly, our study further shows that a pre incubation or co-application of MK 801 prevents HgCl2-induced reduction of cell viability and a disruption of β-tubulin. Conclusions Collectively, our data show that HgCl2-induced toxic effects on central neurons are triggered by an over-activation of NMDA receptors, leading to cytoskeleton instability.

  9. Protection of Hydroxyl Groups as a Trimethylsilyl Ether by1,1,1,3,3,3-Hexamethyldisilazane Promoted by Aspartic Acid as an Efficient Organocatalyst%Protection of Hydroxyl Groups as a Trimethylsilyl Ether by 1,1,1,3,3,3-Hexamethyldisilazane Promoted by Aspartic Acid as an Efficient Organocatalyst

    Institute of Scientific and Technical Information of China (English)

    Arash GHORBANI-CHOGHAMARANI; Masoomeh NOROUZI

    2011-01-01

    A wide variety of alcohols and phenols were protected as trimethylsilyl ethers using 1,1,1,3,3,3-hexamethyl disilazane catalyzed by aspartic acid as a non-toxic, metal-free, and green organocatalyst at room temperature in acetonitrile under mild and heterogeneous conditions. The procedure is operationally simple and the silylated product was obtained in high yield and purity.

  10. Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

    Institute of Scientific and Technical Information of China (English)

    Weiliang Zhao; Dezhi Kang; Yuanxiang Lin

    2008-01-01

    BACKGROUND: Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE: To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY: A computer-based online search was conducted in PUBMED for English language publications containing the key words "brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor" from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria: ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria: duplicated articles.LITERATURE EVALUATION: References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS: After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these

  11. Nanofiltration of L-phenylalanine and L-aspartic acid aqueous solution and its process simulation%苯丙氨酸和天冬氨酸水溶液的纳滤分离及其过程模拟

    Institute of Scientific and Technical Information of China (English)

    王晓琳; 营爱玲

    2001-01-01

    选择苯丙氨酸和天冬氨酸水溶液进行了纳滤分离过程研究,讨论了不同pH下氨基酸的透过特性,并就该体系的浓缩分离进行了模拟计算.%The permeation experiments of aqueous solution of amino acids (L-phenylalanine and L-Aspartic acid) were carried out with nanofiltration membranes. The rejections of phenylalanine and aspartic acid are about 0 and 90%, respectively. The results show that the amino acids having large differences of isoelectric points (pI) can be separated satisfactorily by adjusting the value of pH. Then the concentration and separation process of phenylalanine and aspartic acid solution was simulated. A batchwise concentration and diafiltration system was designed. The results show that it is feasible to separate phenylalanine and aspartic acid in viewpoint of technology.

  12. Effect of mammals’ excretory function on aspartate aminotransferase activity in Glechoma hederacea leaves in conditions of Cd pollution

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    O. M. Vasilyuk

    2014-07-01

    Full Text Available The paper includes analysis of research of Cd impact on the activity of the enzyme of aspartate aminotransferase (AST nitrogen metabolism and the content of water-soluble protein fraction (albumin in Glechoma hederacea L. leaves, which dominated in the research area (in natural floodplain oak forest with Stellaria holostea L.. Cd was introduced in the form of salts of Cd(NO32 in the range of concentrations of: 0.25, 1.25, 2.5 g/m2, equivalent to the inclusion of Cd in 1, 5, 10 doses of MAC. Increase (P < 0.05 in the activity of AST 2.6–3.0 times (with adding Cd salts at a dose of 1 and 5 МAС and albumin content by 37% (with adding Cd salts at a dose of 10 МAС compared to control (the area without Cd pollution and excretory activity of mammals was shown. Using of excreta of some representatives of mammals (for example, Capreolus capreolus L. contributed to reduction of Cd toxic effects and restoring of the functional metabolic activity of AST by 23% (with Cd 1 МAС and by 34% (Cd 5 МAС. It is the evidence of protective function of mammals and their normalization effect at the above concentrations of Cd. Whereas the adding of Cd salts at a dose of 10 МAС led to 3 times’ inhibition of AST activity, the toxic effect of metal by excretory function of mammals was not reduced. Observations revealed the albumin content normalization by 22% in the presence of Cd 1MAC respectively (with the introduction of C. capreolus excreta and to the control level (the area without Cd pollution and excretory activity of mammals with the excreta of Sus scrofa L. in the setting of Cd 10 MAC. It proves the need to use the different mammal species for integrated and comprehensive normalization of ecosystems under conditions of uncontrolled anthropogenic pollution.

  13. Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.

    Science.gov (United States)

    Dong, Xunyan; Zhao, Yue; Zhao, Jianxun; Wang, Xiaoyuan

    2016-06-01

    Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering. PMID:27033538

  14. Secreted aspartic protease cleavage of Candida albicans Msb2 activates Cek1 MAPK signaling affecting biofilm formation and oropharyngeal candidiasis.

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    Sumant Puri

    Full Text Available Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1 phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA, a selective inhibitor of aspartic proteases (Saps. Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ or Sap8 (sap8Δ/Δ resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

  15. Des-Aspartate-Angiotensin I Attenuates Mortality of Mice Exposed to Gamma Radiation via a Novel Mechanism of Action.

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    Hong Wang

    Full Text Available ACE inhibitors and ARBs (angiotensin receptor blockers have been shown to attenuate radiation injuries in animal models of lethal gamma irradiation. These two classes of drug act by curtailing the actions of angiotensin II-linked inflammatory pathways that are up-regulated during gamma radiation in organ systems such as the brain, lung, kidney, and bone marrow. ACE inhibitors inhibit ACE and attenuate the formation of angiotensin II from angiotensin I; ARBs block the angiotensin AT1 receptor and attenuate the actions of angiotensin II that are elicited through the receptor. DAA-I (des-aspartate-angiotensin I, an orally active angiotensin peptide, also attenuates the deleterious actions of angiotensin II. It acts as an agonist on the angiotensin AT1 receptor and elicits responses that oppose those of angiotensn II. Thus, DAA-I was investigated for its anticipated radioprotection in gamma irradiated mice. DAA-I administered orally at 800 nmole/kg/day for 30 days post exposure (6.4 Gy attenuated the death of mice during the 30-day period. The attenuation was blocked by losartan (50 nmole/kg/day, i.p. that was administered sequential to DAA-I administration. This shows that the radioprotection was mediated via the angiotensin AT1 receptor. Furthermore, the radioprotection correlated to an increase in circulating PGE2 of surviving animals, and this suggests that PGE2 is involved in the radioprotection in DAA-I-treated mice. At the hematopoietic level, DAA-I significantly improved two syndromes of myelosuppression (leucopenia and lymphocytopenia, and mice pre-treated with DAA-I prior to gamma irradiation showed significant improvement in the four myelodysplastic syndromes that were investigated, namely leucopenia, lymphocytopenia, monocytopenia and thrombocytopenia. Based on the known ability of PGE2 to attenuate the loss of functional hematopoietic stem and progenitor cells in radiation injury, we hypothesize that PGE2 mediated the action of DAA

  16. Translating the N-methyl-D-aspartate receptor antagonist model of schizophrenia to treatments for cognitive impairment in schizophrenia.

    Science.gov (United States)

    Meltzer, Herbert Y; Rajagopal, Lakshmi; Huang, Mei; Oyamada, Yoshihiro; Kwon, Sunoh; Horiguchi, Masakuni

    2013-11-01

    The N-methyl-D-aspartate receptor (NMDAR) antagonists, phencyclidine (PCP), dizocilpine (MK-801), or ketamine, given subchronically (sc) to rodents and primates, produce prolonged deficits in cognitive function, including novel object recognition (NOR), an analog of human declarative memory, one of the cognitive domains impaired in schizophrenia. Atypical antipsychotic drugs (AAPDs) have been reported to improve declarative memory in some patients with schizophrenia, as well as to ameliorate and prevent the NOR deficit in rodents following scNMDAR antagonist treatment. While the efficacy of AAPDs to improve cognitive impairment in schizophrenia (CIS) is limited, at best, and controversial, single doses of all currently available AAPDs so far tested transiently restore NOR in rodents following scNMDAR antagonist treatment. Typical antipsychotic drugs (APDs), e.g. haloperidol and perphenazine, are ineffective in this rodent model, and may be less effective as treatments of some domains of CIS. Serotonergic mechanisms, including, but not limited to serotonin (5-HT)2A and 5-HT7 antagonism, 5-HT(1A), and GABA(A) agonism, contribute to the efficacy of the AAPDs in the scNMDAR antagonist rodent models, which are relevant to the loss of GABA interneuron/hyperglutamate hypothesis of the etiology of CIS. The ability of sub-effective doses of the atypical APDs to ameliorate NOR in the scNMDAR-treated rodents can be restored by the addition of a sub-effective dose of the 5-HT(1A) partial agonist, tandospirone, or the 5-HT7 antagonist, SB269970. The mGluR2/3 agonist, LY379268, which itself is unable to restore NOR in the scNMDAR-treated rodents, can also restore NOR when given with lurasidone, an AAPD. Enhancing cortical and hippocampal dopamine and acetylcholine efflux, or both, may contribute to the restoration of NOR by the atypical APDs. Importantly, co-administration of lurasidone, tandospirone, or SB269970, with PCP, to rodents, at doses 5-10 fold greater than those

  17. Effect of Terminalia Chebula (Haritaki on Serum Aspartate Aminotransferase, Alanine Aminotransferase in Paracetemol induced liver damage in Wister Albino Rats

    Directory of Open Access Journals (Sweden)

    Tania Yeasmin

    2015-06-01

    Full Text Available Background: Liver plays a major role in detoxification and excretion of many endogenous and exogenous compounds. Any injury may lead to severe liver damage and impairment of liver function. Harbal plants such as Terminalia chebula (Haritaki may have free radical scavenging activity thereby can be used for the prevention and treatment of liver damage. Objective: To observe the effect of Terminalia chebula on paracetamol induced changes of serum aspartate aminotransferase (AST and alanine aminotransferase (ALT in Wister albino rats. Methods: This experimental study was carried out in the Department of Physiology, Dhaka Medical College, Dhaka from January to December’ 2013. Total 44 rats with age 90 to 120 days, weighing between 150 to 200 gm were selected. After acclimatization for 14 days, they were divided into base line control (BC, n=11, paracetamol treated control (PC, n=11,Terminalia chebula pretreated and paracetamol treated (TCP-PCT n=11 and paracetamol pretreated and Terminalia chebula treated group (PCP-TCT, n=11. All groups received basal diet for 21 consecutive days. In addition to basal diet, rats of BC received propylene glycol (2ml/kg body weight, orally and PC received single dose of paracetamol suspension (750mg/kg body weight, orally on 21st day. Rats of TCP-PCT received Terminalia chebula extract (200 mg/kg body weight, orally for 21 consecutive days and paracetamol suspension (750mg/kg body weight, orally on 21st day. Again, rats of PCP-TCT received paracetamol suspension (750mg/kg body weight, orally on the 1st day and Terminalia chebula extract (200 mg/kg body weight orally for 21 consecutive days. All rats were sacrificed on 22nd day and then blood samples were collected. For assessment of liver function serum AST and ALT levels were estimated by using standard laboratory kits. The statistical analysis was done by one way ANOVA and post hoc Bonferroni test as applicable. Results: The mean serum AST and ALT levels were

  18. The squash aspartic proteinase inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family.

    Science.gov (United States)

    Christeller, John T; Farley, Peter C; Marshall, Richelle K; Anandan, Ananda; Wright, Michele M; Newcomb, Richard D; Laing, William A

    2006-12-01

    The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed approximately 30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering approximately 99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family.

  19. The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

    Science.gov (United States)

    Canyuk, B; Focia, P J; Eakin, A E

    2001-03-01

    The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

  20. Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids.

    Science.gov (United States)

    Koponen, S; Keinänen, R; Roivainen, R; Hirvonen, T; Närhi, M; Chan, P H; Koistinaho, J

    1999-01-01

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and

  1. Electrocatalytic oxidation and determination of dopamine at a carbon ionic liquid electrode modified with nafion-L-aspartic acid composite film

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The electrocatalytic oxidation of dopamine(DA)was studied by electrochemical approaches at a carbon ionic liquid electrode(CILE)modified with the composite film of nafion and L-aspartic acid(NL-CILE).The CILE was fabricated by replacing non-conductive organic binders with a room-temperature hydrophobic ionic liquid,1-butyl-3-methyl-imidazolium hexafluorophosphate.The composite film of NL was used as matrix to adsorb DA and catalyze the oxidation of DA in phosphate buffer solution(PBS).The electrochemical re...

  2. Synthesis and characterization of new optically active copoly(amid-imide)s based on N-phthalimido-L-aspartic acid and aromatic diamines

    Institute of Scientific and Technical Information of China (English)

    Khalil; Faghihi; Hamidreza; Alimohammadi

    2010-01-01

    In this article,six new optically active copoly(amide-imide)s(10a-f) were synthesized through the direct polycondensation reaction of N-phthalimido-L-aspartic acid(4) with 1,5-diamino naphthalene(8),3,4-diamino benzophenone(9) in the presence of therphthahc acid(7),fumaric acid(6) and adipic acid(5) as a second diacid in a medium consisting of N-methyl-2-pyrrolidone,triphenyl phosphite, calcium chloride and pyridine.The resulting copolymers were fully characterized by means of FT-IR spectroscopy,elementa...

  3. The glycine transporter GlyT1 controls N-methyl-D-aspartic acid receptor coagonist occupancy in the mouse retina

    OpenAIRE

    Brian T Reed; Sullivan, Steven J.; Tsai, Guochuan; Coyle, Joseph T.; Esguerra, Manuel; Miller, Robert F.

    2009-01-01

    We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1−/+) and compared those results with wild-type (WT) littermate controls (GlyT1+/+). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; ...

  4. Dual effect of beta-amyloid on α7 and α4β2 nicotinic receptors controlling the release of glutamate, aspartate and GABA in rat hippocampus.

    Directory of Open Access Journals (Sweden)

    Elisa Mura

    Full Text Available BACKGROUND: We previously showed that beta-amyloid (Aβ, a peptide considered as relevant to Alzheimer's Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer's Disease pathology, evaluating the effect of Aβ (at different concentrations on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4β2 and α7 subtypes. Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA. METHODOLOGY/FINDINGS: WE USED A DUAL APPROACH: in vivo experiments (microdialysis technique on freely moving rats in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus. Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aβ considered (10 µM in vivo and 100 nM in vitro. In vivo administration of 100 nM Aβ (the lowest concentration considered potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aβ and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4β2 nAChRs selective agonists, respectively elicited the hippocampal release of aspartate, glutamate, and GABA. High Aβ concentrations (100 nM inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aβ concentrations (1 nM and 100 pM selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA. CONCLUSIONS/SIGNIFICANCE: The results reinforce the concept that Aβ has relevant

  5. Aspartic protease inhibitory and nematocidal activity of phenyl-4-(2-phenylhydrazonohexahydrofuro[3,2-c]pyridazin-7-ol (Percival dianhydroosazone

    Directory of Open Access Journals (Sweden)

    El Sayed H. El Ashry

    2014-04-01

    Full Text Available We synthesized Phenyl-4-(2-phenylhydrazonohexahydrofuro[3,2-c]pyridazin-7-ol (compound 3. The structure compound 3 was elucidated with IR, 1H NMR, 13C NMR and EIMS spectra. Compound 3 showed potent inhibitory activity against aspartic proteases, human cathepsin D and Plasmodium falciparum plasmepsin-II with IC50 = 20 μM. Enzyme-inhibitor complexes were predicted to stabilize by electrostatic and hydrophobic interactions between the side chains of amino acid residues at the active center and compound 3. Moreover, compound 3 displayed good nematocidal activity against all developmental stages of C. elegans.

  6. Differential lipid profile and hormonal response in type 2 diabetes by exogenous insulin aspart versus the insulin secretagogue repaglinide, at the same glycemic control

    OpenAIRE

    Chisalita, Simona Ioana; Lindström, Torbjörn; Eson Jennersjö, Pär; Paulsson, Johan; Westermark, Gunilla; Olsson, Anders; Arnqvist, Hans

    2009-01-01

    Our aim was to study, at the same glycemic control, how treatment with either the insulin secretagogue repaglinide or exogenous insulin aspart affects endogenous insulin secretion, plasma insulin and IAPP (islet amyloid polypeptide) levels, GH-IGF (growth hormone-insulin-like growth factor) axis and plasma lipoprotein concentrations in patients with type 2 diabetes. Five patients, age 65.0 +/- A 4.1 years (mean +/- A SE), body weight 82.5 +/- A 5.0 kg, BMI (body mass index) 27.7 +/- A 1.5 kg/...

  7. Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of the glutamate receptor responsive to N-methyl-D-aspartate.

    OpenAIRE

    Schramm, M.; Eimerl, S; Costa, E

    1990-01-01

    The life span of neonatal rat cerebellar granule cells, grown in basal minimal Eagle's medium containing 10% (vol/vol) fetal calf serum, was extended to 21-30 days by weekly supplementation with glucose. Addition of 1% fetal calf serum to the culture at 14 days killed 85% of the cells within 1 hr. This lethal effect could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists dibenzocyclohepteneimine (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). These findi...

  8. Impact of elevated aspartate and alanine aminotransferase on metabolic syndrome and its components among adult people living in Ningxia, China

    Institute of Scientific and Technical Information of China (English)

    Kun-Peng He; Chuan Zhao; Yan Qiang; He-Rong Liu; Nan Chen; Xiu-Juan Tao; Li-Li Chen; Hui Song

    2015-01-01

    Objective: Metabolic syndrome (MS) is a combination of medical disorders that increase the risk for cardiovascular disease and diabetes mellitus. It suggests an association between an elevated serum aminotransferase level and MS. Little data show the relationship between the levels of serum aminotransferase and the incidence of MS in Ningxia, China. Methods: A total of 5415 subjects who received medical health checkups from 2007 to 2009 were enrolled in the study. The participants were interviewed by trained health workers under a structured questionnaire. MS was defined according to the modified ATPIII criteria for Asian Americans by the American Heart Association (AHA-ATP III). Results: The prevalence of elevated aspartate aminotransferase (AST) and ALT (>40 U/L) were 7.1%and 22.2%in males, and 2.1%and 4.8%in females respectively. The prevalence of MS was 32.1%in males and 15.4%in females. The components of MS were significantly more in the group with elevated aminotransferase levels than in the group with normal amino-transferase levels. The odds ratios (95%CI) for elevated AST were 1.90 (1.49, 2.42), 2.59 (2.01, 3.39), 1.68 (1.32, 2.15), and 1.81 (1.36, 2.42) in the adults with abdominal obesity, high serum triglycerides levels, high blood pressure, and high plasma glucose levels respectively. After adjustment for age, the odds ratios (95%CI) for elevated ALT were 3.08 (2.63, 3.61), 4.30 (3.64, 5.08), 1.26 (1.08, 1.48), 2.16 (1.93, 2.65) and 2.38 (1.96, 2.87) in adults with abdominal obesity, high serum tri-glycerides levels, low serum high-density lipoproteincholesterol (HDL-C), high blood pressure, and high plasma glucose levels respectively. The odds ratios (95%CI) for elevated AST were 1.67 (1.06, 2.63), 2.28 (1.46, 3.63), 2.59 (1.59, 4.21) and for elevated ALT 2.02 (1.50, 2.73), 2.68 (1.96, 3.65), 3.94 (2.86, 5.43) for the subjects with 1, 2, and ?3 risk factors after adjustment for age, gender, and BMI. Conclusion: The serum aminotransferase levels were

  9. Arginine–glycine–aspartic acid–polyethylene glycol–polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture

    Directory of Open Access Journals (Sweden)

    Chen Z

    2016-08-01

    Full Text Available Zhanfei Chen,1,* Fen Lian,1,* Xiaoqian Wang,1 Yanling Chen,1,2 Nanhong Tang1,2 1Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 2Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Research Center for Molecular Medicine, Fujian Medical University, Fuzhou, People’s Republic of China *These authors contributed equally to this work Abstract: The polyamidoamine (PAMAM dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine–glycine–aspartic acid (RGD peptide. Our studies demonstrate that RGD–polyethylene glycol (PEG–PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT–MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. Keywords: dendrimer, arginine–glycine–aspartic acid (RGD, liver cell, spheroid culture, ammonia metabolism

  10. Crystal Structure of Cockroach Allergen Bla g 2, an Unusual Zinc Binding Aspartic Protease with a Novel Mode of Self-inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Gustchina, Alla; Li, Mi; Wunschmann, Sabina; Chapman, Martin D.; Pomes, Anna; Wlodawer, Alexander (INDOOR Bio.); (NIH)

    2010-07-19

    The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to {approx}4 {angstrom} and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.

  11. Synthesis and Properties of pH-, Thermo-, and Salt-Sensitive Modified Poly(aspartic acid/Poly(vinyl alcohol IPN Hydrogel and Its Drug Controlled Release

    Directory of Open Access Journals (Sweden)

    Jingqiong Lu

    2015-01-01

    Full Text Available Modified poly(aspartic acid/poly(vinyl alcohol interpenetrating polymer network (KPAsp/PVA IPN hydrogel for drug controlled release was synthesized by a simple one-step method in aqueous system using poly(aspartic acid grafting 3-aminopropyltriethoxysilane (KH-550 and poly(vinyl alcohol (PVA as materials. The hydrogel surface morphology and composition were characterized by Fourier transform infrared spectroscopy (FTIR and scanning electron microscopy (SEM. The thermal stability was analyzed by thermogravimetric analysis (TGA. The swelling properties and pH, temperature, and salt sensitivities of KPAsp, KPAsp/PVA semi-interpenetrating polymer network (semi-IPN, and KPAsp/PVA IPN hydrogels were also investigated. All of the three hydrogels showed ampholytic pH-responsive properties, and swelling behavior was also extremely sensitive to the temperature, ionic strength, and cationic species. Finally, the drug controlled release properties of the three hydrogels were evaluated and results indicated that three hydrogels could control drug release by external surroundings stimuli. The drug controlled release properties of KPAsp/PVA IPN hydrogel are the most outstanding, and the correlative measured release profiles of salicylic acid at 37°C were 32.6 wt% at pH = 1.2 (simulated gastric fluid and 62.5 wt% at pH = 7.4 (simulated intestinal fluid, respectively. These results indicated that KPAsp/PVA IPN hydrogels are a promising carrier system for controlled drug delivery.

  12. Synthesis and characterization of aspartic acid-capped CdS/ZnS quantum dots in reverse micelles and its application to Hg(II) determination

    Energy Technology Data Exchange (ETDEWEB)

    Hosseini, Mohammad Saeid, E-mail: mshosseini1336@yahoo.com; Kamali, Mohsen

    2015-11-15

    In this work, CdS/ZnS quantum dots (QDs) coated with aspartic acid (AsA) were synthesized in reverse micelles. The synthesized QDs were characterized by XRD, TEM, IR and photoluminescence (PL) spectroscopy. It was found that the intensity of CdS/ZnS QDs coated with AsA is much greater than CdS, and CdS/ZnS QDs. The interaction of some heavy metal ions with CdS/ZnS/AsA QDs was investigated at different buffering pH media. Based on the PL quenching of the QDs in the presence of each one of the metal ions, the feasibility of their determinations was examined according to the Stern–Volmer equation. The investigations showed that Hg(II) ions can be easily determined in contaminated atmospheric environments with the detection limit of 0.05 mg m{sup −3}. The results were satisfactorily confirmed by cold vapor atomic absorption spectrometric method. - Highlights: • A new CdS/ZnS quantum dot capped with aspartic acid (DDBA) was prepared. • The prepared QDs benefit from a favorable fluorescence. • Interaction of some metal ions with the QDs was examined according to the Stern–Volmer equation. • The determination of Hg(II) is feasible in the present of many co-existence metal ions. • The method benefits from a high-speed and considerable simplicity for Hg(II) determination.

  13. A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

    Directory of Open Access Journals (Sweden)

    Qing-Xiu Hu

    2012-01-01

    Full Text Available A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession, and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

  14. Corticosterone, cortisol, triglycerides, aspartate aminotransferase and uric acid plasma concentrations during foie gras production in male mule ducks (Anas platyrhynchos × Cairina moschata).

    Science.gov (United States)

    Flament, A; Delleur, V; Poulipoulis, A; Marlier, D

    2012-01-01

    1. Corticosterone, cortisol, triglycerides, aspartate aminotransferase (AST) and uric acid (UA) plasma concentration were measured at 8 (7 days after group housing), 12 (after 7 days of force feeding) and 13 weeks of age (at slaughter after 12 days of force feeding), and 45 min after an adrenocorticotrophic hormone (ACTH) stimulation test at 8 weeks of age in 12 male mule ducks in an on-farm experiment. 2. No significant increase of corticosterone was found during the force-feeding period compared with the concentration after housing. 3. Comparison of corticosterone and cortisol values indicates that cortisol can be considered as a reliable acute stress indicator in future routine examinations. 4. Plasma concentrations of triglycerides and aspartate aminotransferase increased progressively from pre-force feeding period to slaughtering. 5. Plasma concentrations of uric acid increased from the start at 8 weeks of age to the mid-force feeding period but no difference was noticed between the mid-force feeding period and slaughtering. 6. It is concluded that acute stress induced by force-feeding is similar at the beginning and end of the commercial production of foie gras.

  15. Synthesis and Properties of pH-, Thermo-, and Salt-Sensitive Modified Poly(aspartic acid)/Poly(vinyl alcohol) IPN Hydrogel and Its Drug Controlled Release.

    Science.gov (United States)

    Lu, Jingqiong; Li, Yinhui; Hu, Deng; Chen, Xiaoling; Liu, Yongmei; Wang, Liping; Zhao, Yansheng

    2015-01-01

    Modified poly(aspartic acid)/poly(vinyl alcohol) interpenetrating polymer network (KPAsp/PVA IPN) hydrogel for drug controlled release was synthesized by a simple one-step method in aqueous system using poly(aspartic acid) grafting 3-aminopropyltriethoxysilane (KH-550) and poly(vinyl alcohol) (PVA) as materials. The hydrogel surface morphology and composition were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The thermal stability was analyzed by thermogravimetric analysis (TGA). The swelling properties and pH, temperature, and salt sensitivities of KPAsp, KPAsp/PVA semi-interpenetrating polymer network (semi-IPN), and KPAsp/PVA IPN hydrogels were also investigated. All of the three hydrogels showed ampholytic pH-responsive properties, and swelling behavior was also extremely sensitive to the temperature, ionic strength, and cationic species. Finally, the drug controlled release properties of the three hydrogels were evaluated and results indicated that three hydrogels could control drug release by external surroundings stimuli. The drug controlled release properties of KPAsp/PVA IPN hydrogel are the most outstanding, and the correlative measured release profiles of salicylic acid at 37°C were 32.6 wt% at pH = 1.2 (simulated gastric fluid) and 62.5 wt% at pH = 7.4 (simulated intestinal fluid), respectively. These results indicated that KPAsp/PVA IPN hydrogels are a promising carrier system for controlled drug delivery. PMID:26351630

  16. Synthesis of aqueous suspensions of magnetic nanoparticles with the co-precipitation of iron ions in the presence of aspartic acid

    Science.gov (United States)

    Pušnik, Klementina; Goršak, Tanja; Drofenik, Miha; Makovec, Darko

    2016-09-01

    There is increasing demand for the production of large quantities of aqueous suspensions of magnetic iron-oxide nanoparticles. Amino acids are one possible type of inexpensive, nontoxic, and biocompatible molecules that can be used as the surfactants for the preparation of stable suspensions. This preparation can be conducted in a simple, one-step process based on the co-precipitation of Fe3+/Fe2+ ions in the presence of the amino acid. However, the presence of this amino acid changes the mechanism of the magnetic nanoparticles' formation. In this investigation we analyzed the influence of aspartic amino acid (Asp) on the formation of magnetic iron-oxide nanoparticles during the co-precipitation. The process of the nanoparticles' formation was followed using a combination of TEM, x-ray diffractometry, magnetic measurements, in-situ FT-IR spectroscopy, and chemical analysis, and compared with the formation of nanoparticles without the Asp. The Asp forms a coordination complex with the Fe3+ ions, which impedes the formation of the intermediate iron oxyhydroxide phase and suppresses the growth of the final magnetic iron-oxide nanoparticles. Slower reaction kinetics can lead to the formation of nonmagnetic secondary phases. The aspartic-acid-absorbed nanoparticles can be dispersed to form relatively concentrated aqueous suspensions displaying a good colloidal stability at an increased pH.

  17. Nanoparticle carriers based on copolymers of poly(l-aspartic acid co-l-lactide)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine for drug delivery

    Energy Technology Data Exchange (ETDEWEB)

    Han Siyuan; Wang Huan; Liang Xingjie [National Center for Nanoscience and Technology, Laboratory of Nanobiomedicine and Nanosafety, Division of Nanomedicine and Nanobiology (China); Hu Liming, E-mail: huliming@bjut.edu.cn [Beijing University of Technology, College of Life Science and Bioengineering (China); Li Min; Wu Yan, E-mail: wuy@nanoctr.cn [National Center for Nanoscience and Technology, Laboratory of Nanobiomedicine and Nanosafety, Division of Nanomedicine and Nanobiology (China)

    2011-09-15

    A novel poly(l-aspartic) derivative (PAL-DPPE) containing polylactide and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) segments has been successfully synthesized. The chemical structures of the copolymers were confirmed by Fourier-transform infrared spectroscopy (FTIR), NMR ({sup 1}H NMR, {sup 13}C NMR, {sup 31}P NMR), and thermogravimetric analysis (TGA). Fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) confirmed the formation of micelles of the PAL-DPPE copolymers. In order to estimate the feasibility as novel drug carriers, an anti-tumor model drug doxorubicin (DOX) was incorporated into polymeric micelles by double emulsion and nanoprecipitation method. The DOX-loaded micelle size, size distribution, and encapsulation efficiency (EE) were influenced by the feed weight ratio of the copolymer to DOX. In addition, in vitro release experiments of the DOX-loaded PAL-DPPE micelles exhibited that faster release in pH 5.0 than their release in pH 7.4 buffer. The poly(l-aspartic) derivative copolymer was proved to be an available carrier for the preparation of micelles for anti-tumor drug delivery.

  18. Competitive binding of postsynaptic density 95 and Ca2+-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

    Institute of Scientific and Technical Information of China (English)

    Fan-jie MENG; Jun GUO; Bo SONG; Xue-bo YAN; Guang-yi ZHANG

    2004-01-01

    AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca2+-calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10 min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.

  19. Synthesis and characterization of aspartic acid-capped CdS/ZnS quantum dots in reverse micelles and its application to Hg(II) determination

    International Nuclear Information System (INIS)

    In this work, CdS/ZnS quantum dots (QDs) coated with aspartic acid (AsA) were synthesized in reverse micelles. The synthesized QDs were characterized by XRD, TEM, IR and photoluminescence (PL) spectroscopy. It was found that the intensity of CdS/ZnS QDs coated with AsA is much greater than CdS, and CdS/ZnS QDs. The interaction of some heavy metal ions with CdS/ZnS/AsA QDs was investigated at different buffering pH media. Based on the PL quenching of the QDs in the presence of each one of the metal ions, the feasibility of their determinations was examined according to the Stern–Volmer equation. The investigations showed that Hg(II) ions can be easily determined in contaminated atmospheric environments with the detection limit of 0.05 mg m−3. The results were satisfactorily confirmed by cold vapor atomic absorption spectrometric method. - Highlights: • A new CdS/ZnS quantum dot capped with aspartic acid (DDBA) was prepared. • The prepared QDs benefit from a favorable fluorescence. • Interaction of some metal ions with the QDs was examined according to the Stern–Volmer equation. • The determination of Hg(II) is feasible in the present of many co-existence metal ions. • The method benefits from a high-speed and considerable simplicity for Hg(II) determination

  20. Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.).

    Science.gov (United States)

    Williamson, C L; Slocum, R D

    1994-05-01

    We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro. PMID:8029359

  1. root uv-b sensitive Mutants Are Suppressed by Specific Mutations in ASPARTATE AMINOTRANSFERASE2 and by Exogenous Vitamin B6

    Institute of Scientific and Technical Information of China (English)

    Colin D. Leasure; Hong-Yun Tong; Xue-Wen Hou; Amy Shelton; Mike Minton; Raymond Esquerra; Sanja Roje; Hanjo Hellmann; Zheng-Hui He

    2011-01-01

    Vitamin B6 (vitB6)serves as an essential cofactor for more than 140 enzymes. Pyridoxal 5'-phosphate (PLP),active cofactor form of vitB6, can be photolytically destroyed by trace amounts of ultraviolet-B (UV-B). How sun-exposed organisms cope with PLP photosensitivity and modulate vitB6 homeostasis is currently unknown. We previously reported on two Arabidopsis mutants, rusl and rus2, that are hypersensitive to trace amounts of UV-B light. We performed mu-tagenesis screens for second-site suppressors of the rus mutant phenotype and identified mutations in the ASPARTATE AMINOTRANSFERASE2 (ASP2)gene. ASP2 encodes for cytosolic aspartate aminotransferase (AAT), a PLP-dependent en-zyme that plays a key role in carbon and nitrogen metabolism. Genetic analyses have shown that specific amino acid substitutions in ASP2 override the phenotypes of rusl and rus2 single mutants as well as rusl rus2 double mutant. These substitutions, all shown to reside at specific positions in the PLP-binding pocket, resulted in no PLP binding. Additional asp2 mutants that abolish AAT enzymatic activity, but which alter amino acids outside of the PLP-binding pocket, fail to suppress the rus phenotype. Furthermore, exogenously adding vitB6 in growth media can rescue both rusl and rus2. Our data suggest that AAT plays a role in vitB6 homeostasis in Arabidopsis.

  2. Effect of aspartame and protein, administered in phenylalanine-equivalent doses, on plasma neutral amino acids, aspartate, insulin and glucose in man.

    Science.gov (United States)

    Møller, S E

    1991-05-01

    Six human males each received 0.56 g phenylalanine (Phe) in the form of 1.0 g aspartame or 12.2 g bovine albumin in 200 ml water or water alone. Venous blood samples collected before consumption and during the following 4 hr were assayed for plasma levels of large, neutral amino acids (LNAA), aspartate, insulin and glucose. The area under the curve for plasma Phe was 40% greater, although not significant, after aspartame compared with albumin intake. The indicated increased clearance rate of plasma Phe after albumin may be caused by the significant increase of insulin, on which aspartame had no effect. There was a significant main effect of aspartame for plasma tyrosine but not for tryptophan, valine, isoleucine or leucine. Plasma aspartate was significantly increased at 0.25 hr after the aspartame intake. The percentage Phe/LNAA decreased slightly in response to albumin but increased 55% after aspartame and remained significantly increased for 2 hr. Tyrosine/LNAA increased and tryptophan/LNAA decreased modestly after aspartame intake. The study showed that the intake of aspartame in a not unrealistically high dose produced a marked and persistent increase of the availability of Phe to the brain, which was not observed after protein intake. The study indicated, furthermore, that Phe was cleared faster from the plasma after consumption of protein compared with aspartame. PMID:1946186

  3. 聚天冬氨酸阻垢剂的制备及性能评价%Preparation and Performance Evaluation of Poly-aspartic Acid Scale Inhibitor

    Institute of Scientific and Technical Information of China (English)

    杨红丽; 姬彩云

    2015-01-01

    Poly-aspartic acid has good scale inhibition effect. Using maleic anhydride and ammonium chloride, poly-aspartic acid was prepared by hot-polymerization. And the optimum reaction conditions were obtained by or-thogonal experiment as follows:molar ratio of maleic anhydride/ammonium chloride was 1∶1. 2,reaction teampra-ture was 140℃,and reaction time was 5h.%聚天冬氨酸具有较好的阻垢效果。采用热引发聚合,以马来酸酐、氯化铵为原料,制备了绿色阻垢剂聚天冬氨酸,通过正交实验确定了最佳合成条件,并对其进行了性能评价。最佳合成条件为:马来酸酐和氯化铵的摩尔比为1∶1.2,反应温度为140℃,反应时间为5 h。

  4. Detection of Serine Octamer by Desorption Electrospray Ionization Mass Spectrometry in Resultant Mixture of Aspartic Acid Exposed to Sunshine Under Natural Conditions

    Institute of Scientific and Technical Information of China (English)

    CHEN Huan-wen; LI Ming; JIN Wei; JIN Qin-han; ZHENG Jian

    2007-01-01

    Serine, one of the nonessential amino acids, is of principal interest because of its capability to form magic-number ionic clusters, which provide a remarkable preference for homochirality. With L-aspartic acid as the precursor,this study provides experimental evidence for serine formation in weak acidified aqueous solutions in the presence of iron, with exposure to sunlight, which simulates the natural conditions of the prebiotic aqueous environment. The resultant mixture is directly analyzed via desorption electrospray ionization mass spectrometry( DESI-MS), without any sample preseparation. The serine monomer is successfully detected as protonated molecules, giving a peak at m/z 106, which is confirmed by the MS/MS fragments. Protonated serine octamer(m/z 841) is also observed with significant abundance in the MS spectra and is confirmed by the MS/MS data, which shows the formation of the serine octamer by a synthesized serine in the resultant mixture. It is also found that the serine octamer yielded equivalent abundance in the DESI mass spectra in a wide pH range(pH=1-5), and that existence of ferrous salt and sunshine are essential for the conversion of aspartic acid to serine in the acidic water solution.

  5. Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

    Directory of Open Access Journals (Sweden)

    Xu Chun-Ling

    2011-11-01

    Full Text Available Abstract Background Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up. Case presentation A 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen. Conclusions Persistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year

  6. Controlling N-methyl-D-aspartate receptor subunit 1 with calcitonin gene related peptide after cerebral ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Jixiang Cui; Peng Qu; Chunping Qiao

    2006-01-01

    BACKGROUND: Activation of N-methyl-D-aspartate receptor(NMDAR)is a key link of exitotoxictiy at the phase of cerebral ischemic injury.Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors,over-excitation can cause neuronal apoptosis.Calcitonin gene related peptide has a strongly biological activity.On one hand,it can play the protective effect through inhibiting the expression of NMDAR1 mRNA;on the other hand,it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide.OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion(I/R).DESIGN: Randomized controlled animal study.SETTING: China Medical University.MATERIALS: A total of 216 healthy male Wistar rats,general grade,weighing 250-280 g,were selected in this study.Twelve rats were randomly selected to regard as control group;meanwhile,other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO)models.The main reagents were detailed as follows:calcitonin gene related peptide (Sigma Company);calcitonin gene related peptide kit (Boster Company);antibodyⅠ,Ⅱand antibody β-actin Ⅰ,Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company,USA)METHODS:The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ①Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1,2 and 3 after wakefulness,the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group.All rats in the both groups were observed at five time points

  7. 苯丙氨酸和天冬氨酸水溶液的纳滤分离过程研究%The Nanofiltration Process of Aqueous Solution of Phenylalanine and Aspartic Acid

    Institute of Scientific and Technical Information of China (English)

    营爱玲; 王晓琳

    2001-01-01

    The permeation experiments on the aqueous solution of phenylalanine and aspartic acid were carried out with nanofiltration membranes. Results show that amino acids with different isoelectric points (pI) can be separated satisfactorily by adjusting the pH values. The concentration and separation process of phenylalanine and aspartic acid solution was also simulated.%选择L-苯丙氨酸和L-天冬氨酸水溶液进行了纳滤分离过程研究,讨论了不同pH下氨基酸的透过特性,并进行了模拟计算.

  8. Effect of propofol on the reactivity of acetylcholinesterase, N-methyl-D-aspartate receptors, and gamma-aminobutyric acid receptors in the hippocampus of aged rats after chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Gang Chen; Jiangbei Cao; Weidong Mi

    2011-01-01

    We induced ischemic brain injury in aging rats to examine the effects of varying doses of propofol on hippocampal activities of acetylcholinesterase, N-methyl-D-aspartate receptors, and γ-aminobutyric acid receptors. Propofol exhibited no obvious impact on acetylcholinesterase activity, but directly activated the γ-aminobutyric acid receptor. The neuroprotective function of propofol on the hippocampus of aging rats following cerebral ischemic injury may be related to altered activities of γ-aminobutyric acid receptors and N-methyl-D-aspartate receptors.

  9. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    OpenAIRE

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrat...

  10. Synthesis and characterization of a series of diarylguanidines that are noncompetitive N-methyl-D-aspartate receptor antagonists with neuroprotective properties

    Energy Technology Data Exchange (ETDEWEB)

    Keana, J.F.W.; McBurney, R.N.; Scherz, M.W.; Fischer, J.B.; Hamilton, P.N.; Smith, S.M.; Server, A.C.; Finkbeiner, S.; Stevens, C.F.; Jahr, C.; Weber, E. (Univ. of Oregon, Eugene (USA))

    1989-07-01

    Four diarylguanidine derivatives were synthesized. These compounds were found to displace, at submicromolar concentrations, {sup 3}H-labeled 1-(1-(2-thienyl)cyclohexyl)piperidine and (+)-({sup 3}H)MK-801 from phencyclidine receptors in brain membrane preparations. In electrophysiological experiments the diarylguanidines blocked N-methyl-D-aspartate (NMDA)-activated ion channels. These dairylguanidines also protected rat hippocampal neurons in vitro from glutamate-induced cell death. The results show that some diarylguanidines are noncompetitive antagonists of NMDA receptor-mediated responses and have the neuroprotective property that is commonly associated with blockers of the NMDA receptor-gated cation channel. Diarylguanidines are structurally unrelated to known blockers of NMDA channels and, therefore, represent a new compound series for the development of neuroprotective agents with therapeutic value in patients suffering from stroke, from brain or spinal cord trauma, from hypoglycemia, and possibly from brain ischemia due to heart attack.

  11. The concentration of N-acetyl aspartate, creatine + phosphocreatine, and choline in different parts of the brain in adulthood and senium

    DEFF Research Database (Denmark)

    Christiansen, P; Toft, P; Larsson, H B;

    1993-01-01

    The fully relaxed water signal was used as an internal standard in a STEAM experiment to calculate the concentrations of the metabolites: N-acetyl aspartate (NAA), creatine + phosphocreatine (Cr + PCr), and choline (Cho) containing compounds in four different parts of the brain in two age groups....... In the younger age group, the concentration of NAA was significantly higher in the occipital part than in the other three parts of the brain. No significant regional variation was found for any other metabolite concentration. There was a significantly higher concentration of NAA in the occipital part...... of the brain in the younger age group compared to the older one. No significant regional or age dependent variation was found concerning the T1 and T2 relaxation times....

  12. The relaxing effect of perivascular tissue on porcine retinal arterioles in vitro is mimicked by N-methyl-D-aspartate and is blocked by prostaglandin synthesis inhibition

    DEFF Research Database (Denmark)

    Holmgaard, Kim; Aalkjaer, Christian; Lambert, John D C;

    2007-01-01

    PURPOSE: Retinal hyperperfusion resulting from disturbances in the regulation of arteriolar tone is involved in the pathophysiology of a variety of retinal diseases. The mechanisms underlying this regulation of tone involve cellular components in both the vascular wall and the perivascular tissue....... However, previous in vitro studies of the influence of perivascular retinal tissue on retinal tone regulation have been hampered by the release of an endogenous relaxing factor that renders the arteriole insensitive to vasoconstrictors. The purpose of the present study was to test whether N......-methyl-D-aspartate (NMDA) and gamma-amino butyric acid (GABA) receptors, and a cyclooxygenase (COX) product influence this effect of perivascular retinal tissue in vitro. METHODS: Porcine retinal arterioles were mounted in a wire myograph for isometric force measurements. The contractile effect of the prostaglandin...

  13. Ethanol neurobehavioral teratogenesis and the role of the hippocampal glutamate-N-methyl-D-aspartate receptor-nitric oxide synthase system.

    Science.gov (United States)

    Kimura, K A; Reynolds, J N; Brien, J F

    2000-01-01

    The purpose of this review is to evaluate a proposed mechanism for ethanol neurobehavioral teratogenesis in the hippocampus, involving suppression of the glutamate-N-methyl-D-aspartate (NMDA) receptor-nitric oxide synthase (NOS) system. It is postulated that suppression of this signal transduction system in the fetus by chronic maternal consumption of ethanol plays a key role in hippocampal dysmorphology and dysfunction in postnatal life. This mechanism is evaluated critically based on the current literature and our research findings. In view of the apparent time course for loss of CA1 pyramidal cells in the hippocampus produced by chronic prenatal ethanol exposure that manifests in early postnatal life, it is proposed that therapeutic intervention, which targets the glutamate-NMDA receptor-NOS system, may prevent or lessen the magnitude of postnatal hippocampal dysfunction. PMID:11106855

  14. Effect of N-methyl-aspartate and Betaine on Growth Performance and Correlation Between Growth Hormone, Growth Performance and Carcass Composition in Finishing Pigs

    Institute of Scientific and Technical Information of China (English)

    X(U) Zi-rong; FENG Jie; ZOU Xiao-ting

    2002-01-01

    Ninety finishing pigs were selected to study the effect of N-methyl-aspartate and betaine on the internal growth hormone level in the serum and the correlation between the growth hormone level, growth performance and carcass characteristic of finishing pig. The study showed that the two matters could improve pig growth and carcass composition significantly. The correlation analyses indicated that the growth hormone and IGF-I have a positive correlation with the growth rate. Carcass lean ratio, longissimus dorsi area, serum free fatty acid and lipase activity have a negative correlation with the feed conversion ratio, carcass fat ratio and urine nitrogen. But the growth hormone is more effective than IGF-I (P< 0.01 ). The results implicated that both the two matters may act through growth hormone axis(growth hormone - IGF-I) to manipulate pig growth.

  15. Effect of gamma irradiation on the activity of alanine and aspartate transaminases in subcellular fractions of the brain and heart in white rats

    Energy Technology Data Exchange (ETDEWEB)

    Plenin, A.E.

    1973-01-01

    In experiments on rats, the activity of alanine (I) and aspartate transaminases (II) was studied in homogenates and subcellular fractions of the brain and myocardium under normal conditions and for 30 days after ..gamma.. irradiation at 40 rads. The activity of II in brain homogenates increased 1 hour after irradiation but decreased by 20 percent on day 3; it decreased again on days 7 and 15. The activity of brain I increased after 1 hour and 3 days but then returned to normal. The activity of I in heart homogenates increased in all the periods after irradiation. The subcellular fractions exhibited phase changes in the activity of the enzymes. These changes were different in nature from those observed after X and ..gamma.. irradiation at the same dose.

  16. Aspartate aminotransferase catalyzed oxygen exchange with solvent from oxygen-18-enriched alpha-ketoglutarate: Evidence for slow exchange of enzyme-bound water

    Energy Technology Data Exchange (ETDEWEB)

    McLeish, M.J.; Julin, D.A.; Kirsch, J.F. (Univ. of California, Berkeley (USA))

    1989-05-02

    Partitioning of the ketimine (or ketimine + quinonoid) intermediate(s) in the mitochondrial aspartate aminotransferase reactions was investigated by following the rates of loss of {sup 18}O from carbonyl-{sup 18}O-enriched alpha-ketoglutarate together with the rate of L-glutamate formation. The ratio of these rate constants was found to equal 1 at 10{degree}C, implying that the above intermediate(s) face(s) equal barriers with respect to the forward and reverse reactions. This partition ratio of 1 together with that measured from the alpha-amino acid side of the reaction suggests that the rate constant for exchange of alpha-ketoglutarate-derived H{sub 2}(18)O from the ketimine (or ketimine + quinonoid) form(s) of the enzyme with solvent is comparable with that for kcat.

  17. Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

    Science.gov (United States)

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    2016-01-01

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. PMID:27621619

  18. Synthesis, radiolabeling and evaluation of novel amine guanidine derivatives as potential positron emission tomography tracers for the ion channel of the N-methyl-d-aspartate receptor.

    Science.gov (United States)

    Klein, Pieter J; Chomet, Marion; Metaxas, Athanasios; Christiaans, Johannes A M; Kooijman, Esther; Schuit, Robert C; Lammertsma, Adriaan A; van Berckel, Bart N M; Windhorst, Albert D

    2016-08-01

    The N-Methyl-d-Aspartate receptor (NMDAR) is involved in many neurological and psychiatric disorders including Alzheimer's disease and schizophrenia. The aim of this study was to develop a positron emission tomography (PET) ligand to assess the bio-availability of the NMDAR ion channel in vivo. A series of tri-N-substituted diarylguanidines was synthesized and their in vitro binding affinities for the NMDAR ion channel assessed in rat forebrain membrane fractions. Compounds 21, 23 and 26 were radiolabeled with either carbon-11 or fluorine-18 and ex vivo biodistribution and metabolite studies were performed in Wistar rats. Biodistribution studies showed high uptake especially in prefrontal cortex and lowest uptake in cerebellum. Pre-treatment with MK-801, however, did not decrease uptake of the radiolabeled ligands. In addition, all three ligands showed fast metabolism. PMID:27128179

  19. Arginine–glycine–aspartic acid–polyethylene glycol–polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture

    Science.gov (United States)

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    2016-01-01

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine–glycine–aspartic acid (RGD) peptide. Our studies demonstrate that RGD–polyethylene glycol (PEG)–PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT–MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. PMID:27621619

  20. Construction and expression of aspartic protease from Onchocerca volvulus* as ompA fusion protein in a mutant strain of Salmonella typhimurium

    Directory of Open Access Journals (Sweden)

    Jolodar Abbas

    2002-01-01

    Full Text Available Two constructions in pHS164 vector were designed to permit expression of OV7A and OV4A inserts encoding the N-terminal and C-terminal portion of an aspartic protease from Onchocerca volvulus, respectively. A novel 39 kD protein ompA-OV7A fusion protein was stably expressed as ompA fusion in a modified strain of Salmonella typhimurium strain SL5000 and E.coli strain JM109. Expression of the fusion protein in bacterial strains harboring the constructs were evaluated by western blotting. E.coli and Salmonella lysates were fractionated by 10% SDS-PAGE gel and then immobilized to nitrocellulose membrane by electroblotting. Primary polyclonal antibody generated in rats against the GST-OV7A fusion protein was used in the Western blots. It remains to be seen whether the fusion protein expressed in vivo will promote effective immune response.

  1. Behavioral and cognitive effects of the N-methyl-D-aspartate receptor co-agonist D-serine in healthy humans: initial findings.

    Science.gov (United States)

    Levin, Raz; Dor-Abarbanel, Adi Ein; Edelman, Shany; Durrant, Andrea R; Hashimoto, Kenji; Javitt, Daniel C; Heresco-Levy, Uriel

    2015-02-01

    The efficacy of compounds having agonistic activity at the glycine site associated with the N-methyl-D-aspartate receptor (NMDAR) is presently assessed in psychiatric disorders. In contrast to NMDAR antagonists, the neuropsychiatric effects of NMDAR agonists in the healthy human organism are not known. We studied neuropsychiatric and neurochemical effects of the NMDAR-glycine site obligatory co-agonist d-serine (DSR) in healthy subjects using a randomized, controlled crossover challenge design including a baseline assessment day and two DSR/placebo administration days. Thirty-five subjects aged 23-29 years participated in the study and received a 2.1 g orally administered DSR dose. The main outcome measures were the changes in scores of mood-related Visual Analogue Scale (VAS), Continuous Performance Test-Identical Pairs (CPT-IP), and Rey Auditory Verbal Learning Test (RAVLT). DSR acute administration: (1) was well tolerated and resulted at 2 h in ≥ 200 times increase in DSR serum levels; (2) elicited reduced VAS-measured depression and anxiety feelings; (3) improved attention and vigilance as measured by CPT-IP D-prime score; (4) preferentially improved performance in RAVLT list 7 reflecting ability to retain information over interference; (5) had significant but nonspecific effects on Category Fluency and Benton Visual Retention tests; and (6) did not affect glycine and glutamate serum levels. These data indicate that in healthy subjects, DSR reduces subjective feelings of sadness and anxiety and has procognitive effects that are overall opposed to the known effects of NMDAR antagonists. The findings are relevant to translational research of NMDAR function and the development of NMDAR-glycine site treatments for specific psychiatric entities. ClinicalTrials.gov: Behavioral and Cognitive Effects of the N-methyl-D-aspartate Receptor (NMDAR) Co-agonist D-serine in Healthy Humans; http://www.clinicaltrials.gov/ct2/show/NCT02051426?term=NCT02051426&rank=1; NCT

  2. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    Science.gov (United States)

    Yan, H G; Tsai, M D

    1991-06-01

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

    NARCIS (Netherlands)

    De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.

    2006-01-01

    It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the n

  4. Anti-N-methyl-d-aspartate receptor encephalitis in a patient with a 7-year history of being diagnosed as schizophrenia: complexities in diagnosis and treatment

    Directory of Open Access Journals (Sweden)

    Huang C

    2015-06-01

    Full Text Available Chaohua Huang,1,2,4,* Yukun Kang,1,* Bo Zhang,1 Bin Li,1 Changjian Qiu,1 Shanming Liu,1 Hongyan Ren,1,2 Yanchun Yang,1 Xiehe Liu,1 Tao Li,1–3 Wanjun Guo1,21Mental Health Center, West China Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2State Key Laboratory of Biotherapy, Psychiatric Laboratory, West China Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 3Mental Health Education Center, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 4Mental Health Center, Affiliated Hospital of Luzhou Medical College, Luzhou, People’s Republic of China*These authors contributed equally to this workAbstract: Anti-N-methyl-d-aspartate receptor (NMDAR encephalitis is a form of autoimmune encephalitis associated with antibodies against the NR1 subunits of NMDARs. Although new-onset acute prominent psychotic syndromes in patients with NMDAR encephalitis have been well documented, there is a lack of case studies on differential diagnosis and treatment of anti-NMDAR encephalitis after a long-term diagnostic history of functional psychotic disorders. The present study reports an unusual case of anti-NMDAR encephalitis. The patient had been diagnosed with schizophrenia 7 years earlier, and was currently hospitalized for acute-onset psychiatric symptoms. The diagnosis became unclear when the initial psychosis was confounded with considerations of other neurotoxicities (such as neuroleptic malignant syndrome. Finally, identification of specific immunoglobulin G NR1 autoantibodies in the cerebrospinal fluid and greater effectiveness of immunotherapy over antipsychotics alone (which has been well documented in anti-NMDAR encephalitis indicated the diagnosis of anti-NMDAR encephalitis in this case. Based on the available evidence, however, the relationship between the newly diagnosed anti-NMDAR encephalitis and the seemingly clear, long-term history of schizophrenia in the preceding 7

  5. Toxicity assessment of repeated intravenous injections of arginine–glycine–aspartic acid peptide conjugated CdSeTe/ZnS quantum dots in mice

    Directory of Open Access Journals (Sweden)

    Wang YW

    2014-10-01

    Full Text Available You-Wei Wang, Kai Yang, Hong Tang, Dan Chen, Yun-Long Bai Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China Background: Nanotechnology-based near-infrared quantum dots (NIR QDs have many excellent optical properties, such as high fluorescence intensity, good fluorescence stability, and strong tissue-penetrating ability. Integrin αvß3 is highly and specifically expressed in tumor angiogenic vessel endothelial cells of almost all carcinomas. Recent studies have shown that NIR QDs linked to peptides containing the arginine–glycine–aspartic acid (RGD sequence (NIR QDs-RGD can specifically target integrin αvß3 expressed in endothelial cells of tumor angiogenic vessels in vivo, and they offer great potential for early cancer diagnosis, in vivo tumor imaging, and tumor individualized therapy. However, the toxicity profile of NIR QDs-RGD has not been reported. This study was conducted to investigate the toxicity of NIR QDs-RGD when intravenously administered to mice singly and repeatedly at the dose required for successful tumor imaging in vivo.Materials and methods: A NIR QDs-RGD probe was prepared by linking NIR QDs with the maximum emission wavelength of 800 nm (QD800 to the RGD peptide (QD800-RGD. QD800-RGD was intravenously injected to BALB/C mice once or twice (200 pmol equivalent of QD800 for each injection. phosphate-buffered saline solution was used as control. Fourteen days postinjection, toxicity tests were performed, including complete blood count (white blood cell, red blood cell, hemoglobin, platelets, lymphocytes, and neutrophils and serum biochemical analysis (total protein, albumin, albumin/globulin, aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen. The coefficients of liver, spleen, kidney, and lung weight to body weight were measured, as well as their oxidation and antioxidation indicators, including

  6. Approach to the Management of Pediatric-Onset Anti-N-Methyl-d-Aspartate (Anti-NMDA) Receptor Encephalitis: A Case Series.

    Science.gov (United States)

    Brenton, J Nicholas; Kim, Joshua; Schwartz, Richard H

    2016-08-01

    Anti-N-methyl-d-aspartate (anti-NMDA) receptor encephalitis is a treatable cause of autoimmune encephalitis. It remains unclear if the natural history of this disease is altered by choice of acute therapy or the employment of chronic immunotherapy. Chart review was undertaken for pediatric patients diagnosed with anti-NMDA receptor encephalitis. Data obtained included patient demographics, disease manifestations, treatment course, and clinical outcomes. Ten patients with anti-NMDA receptor encephalitis were identified. All patients were treated with immunotherapy in the acute period, and all patients experienced good recovery. Neurologic relapse did not occur in any patient. All patients received varied forms of chronic immunosuppression to prevent relapses. Complications of chronic immunotherapy occurred in 50% of patients. The benefits of chronic immunotherapy and the duration of use should be carefully weighed against the risks. Complications from immunotherapy are not uncommon and can be serious. Clinical trials assessing the benefit of long-term immunotherapy in this population are needed. PMID:27121044

  7. Efficacy and safety of biphasic insulin aspart and biphasic insulin lispro mix in patients with type 2 diabetes: A review of the literature.

    Science.gov (United States)

    Kumar, Ajay

    2016-01-01

    Type 2 diabetes (T2D) represents an escalating burden worldwide, particularly in China and India. Compared with Caucasians, Asian people with diabetes have lower body mass index, increased visceral adiposity, and postprandial glucose (PPG)/insulin resistance. Since postprandial hyperglycemia contributes significantly to total glycemic burden and is associated with heightened cardiovascular risk, targeting PPG early in T2D is paramount. Premixed insulin regimens are widely used in Asia due to their convenience and effectiveness. Data from randomized controlled trials and observational studies comparing efficacy and safety of biphasic insulin aspart 30 (BIAsp 30) with biphasic insulin lispro mix (LM 25/50) and versus other insulin therapies or oral antidiabetic drugs (OADs) in T2D demonstrated that BIAsp 30 and LM 25/50 were associated with similar or greater improvements in glycemic control versus comparator regimens, such as basal-bolus insulin, in insulin-naÏve, and prior insulin users. Studies directly comparing BIAsp 30 and LM 25 provided conflicting glycemic control results. Safety data generally showed increased hypoglycemia and weight gain with premixed insulins versus basal-bolus insulin or OADs. However, large observational trials documented improvements in glycated hemoglobin, PPG, and hypoglycemia with BIAsp 30 in multi-ethnic patient populations. In summary, this literature review demonstrates that premixed insulin regimens are an appropriate and effective treatment choice in T2D. PMID:27186543

  8. Changes in synaptic and extrasynaptic N-methyl-D-aspartate receptor-mediated currents at early-stage epileptogenesis in adult mice

    Institute of Scientific and Technical Information of China (English)

    Juegang Ju; Sheng-tian Li

    2011-01-01

    Previous reports have shown that N-methyl-D-aspartate (NMDA) receptors are extensively involved in epilepsy genesis and recurrence.Recent studies have shown that synaptic and extrasynaptic NMDA receptors play different, or even opposing, roles in various signaling pathways, including synaptic plasticity and neuronal death.The present study analyzed changes in synaptic and extrasynaptic NMDA receptor-mediated currents during epilepsy onset.Mouse models of lithium chloride pilocarpine-induced epilepsy were established, and hippocampal slices were prepared at 24 hours after the onset of status epilepticus.Synaptic and extrasynaptic NMDA receptor-mediated excitatory post-synaptic currents (NMDA-EPSCs) were recorded in CA1 pyramidal neurons by whole-cell patch clamp technique.Results demonstrated no significant difference in rise and delay time of synaptic NMDA-EPSCs compared with normal neurons.Peak amplitude, area-to-peak ratio,and rising time of extrasynaptic NMDA-EPSCs remained unchanged, but decay of extrasynaptic NMDA-EPSCs was faster than that of normal neurons.These results suggest that extrasynaptic NMDA receptors play a role in epileptogenesis.

  9. Water-soluble lipopolymer delivery of N-methyl-D-aspartic acid receptor 2B siRNA relieves chronic neuropathic pain in rats

    Institute of Scientific and Technical Information of China (English)

    Jianhua Lu; Yuanxiang Tao; Xue Yang; Weifeng Tu; Hao Chen; Jiaxiang Xiong; Chungui Hu

    2011-01-01

    Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B (NR2B) overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA (siRNA) can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. However, an efficient and safe vector for NR2B siRNA has not been discovered. This study shows that a water soluble lipopolymer (WSLP) comprised of low molecular weight polyethyleneimine (PEI) and cholesterol can deliver siRNA targeting NR2B for the treatment of neuropathic pain. Results show that intrathecal injection of WSLP/siRNA complexes for 3 days inhibit NR2B gene expression with reductions in mRNA and protein levels by 59% and 54%, respectively, compared with control rats (P < 0.01). Injection of WSLP complexed with scrambled siRNA, or PEI with siRNA did not show this inhibitory effect. Moreover, injection of WSLP/siRNA complexes significantly relieved neuropathic pain at 3, 7, 12, and 21 days, while injection of WSLP with scrambled siRNA or PEI with siRNA did not. These results demonstrate that WSLP can efficiently deliver siRNA targeting NR2B in vivo and relieve neuropathic pain.

  10. Hydrothermal Synthesis and Structure Analysis of a Chromium Complex [Cr(aea)]2(OH)2(aea=N-(2-Aminoethyl)aspartate)

    Institute of Scientific and Technical Information of China (English)

    DANG Dong-Bin; JIN Ya-Nan; BAI Yan; PAN Xiao-Jing; WANG Jing-Ping

    2008-01-01

    A dimeric chromium compound has been synthesized by hydrothermal method and characterized by IR,EPR spectra and TG-DTA technique.The title compound [Cr(aea)]2(OH)2(aea=N-(2-aminoethyl)aspartate)crystallizes in monoclinic,space group P21/c with a=8.9393(10),b=6.7198(7),c=14.6791(16)(A),β=91.0580(10)°,V=881.63(17)(A)3,Z=2,Mr=486.34,Dc=1.832g/cm3,F(000)=500,μ=1.296 mm-1,R=0.0271 and wR=0.0756.Single-crystal X-ray structural analysis indicates that the title compound consists of two Cr(aca)units linked by two bridging hydroxyl groups.The geometry around each chromium center is approximately octahedral and completed by two hydroxyls and a quadridentate ligand which is formed when ethylenediamine reacts with maleie acid.

  11. N-methyl-D-aspartate receptor expression in the spinal dorsal horn of a rat model of formalin-induced inflammatory pain following intrathecal injection of butorphanol

    Institute of Scientific and Technical Information of China (English)

    Yichun Wang; Yuan Zhang; Qulian Guo; Xiaohong Liu; Mingde Wang; Hui Luo

    2010-01-01

    Clinical and animal experiments have proved that intrathecal injection of butorphanol has an analgesic effect. However, whether the analgesic effect is associated with activation of the N-methyl-D-aspartate (NMDA) receptor remains unclear. This study presumed that intrathecal injection of butorphanol has an analgesic effect on formalin-induced inflammatory pain in rats, and its analgesic effect is associated with inhibition of NMDA receptors. Concurrently, ketamine was injected into the intrathecal space, which is a non-competitive NMDA receptor antagonist, to determine the analgesic mechanism of butorphanol. The total reflection time in phase 1 and phase 2 of rat hind paws carding action was reduced when the butorphanol dose was increased to 25 μg,or a low dose of butorphanol was combined with ketamine. Intrathecal injection of a high dose of butorphanol alone or a Iow dose of butorphanol combined with ketamine can remarkably reduce NMDA receptor expression in the L5 spinal dorsal horn of formalin-induced pain rats. The results suggest that intrathecal injection of butorphanol has analgesic effects on formalin-induced inflammatory pain, and remarkably reduces NMDA receptor expression in the rat spinal dorsal horn.Ketamine strengthens this analgesic effect. The analgesic mechanism of intrathecal injection of butorphanol is associated with inhibition of NMDA receptor activation.

  12. A serotonin-1A receptor agonist and an N-methyl-D-aspartate receptor antagonist oppose each others effects in a genetic rat epilepsy model.

    Science.gov (United States)

    Filakovszky, J; Gerber, K; Bagdy, G

    1999-02-12

    The WAG/RIJ rats exhibit spontaneously occurring spike-wave discharges (SWD) accompanied by behavioural phenomena, with characteristics similar to the human absence type epilepsy. To study the mechanisms involved in this type of epileptiform activity we investigated the effects of the serotonin-1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the N-methyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate (MK-801). Intracerebroventricular (i.c.v.) injection of 8-OH-DPAT caused marked, dose dependent increase, MK-801 a decrease in the cumulative duration and number of spike-wave discharges. Pretreatment with MK-801 (10 microg/rat i.c.v.) abolished the increase caused by 8-OH-DPAT (20 microg/rat i.c.v.), but the decrease in SWD to MK-801 was counterbalanced by 8-OH-DPAT. These data provide evidence for an interaction of glutamatergic and serotonergic mechanisms in the triggering and maintenance of epileptic activity in this genetic model of absence epilepsy.

  13. Aspartate-Based CXCR4 Chemokine Receptor Binding of Cross-Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes.

    Science.gov (United States)

    Maples, Randall D; Cain, Amy N; Burke, Benjamin P; Silversides, Jon D; Mewis, Ryan E; D'huys, Thomas; Schols, Dominique; Linder, Douglas P; Archibald, Stephen J; Hubin, Timothy J

    2016-08-26

    The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists. PMID:27458983

  14. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    Science.gov (United States)

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (pbenchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology. PMID:25049573

  15. Synthesis and characterization of novel water-soluble polyamide based on spermine and aspartic acid as a potential gene delivery vehicle

    Directory of Open Access Journals (Sweden)

    2008-05-01

    Full Text Available We developed a novel and convenient method for the synthesis of a potentially safe non-viral gene delivery vehicle based on the cationic block copolymer of spermine and aspartic acid (ASSP and coupled it with polyethylene glycol (PEG. The copolymer ASSP was prepared by direct polycondensation in the ionic liquid, butylmethylimidazolium hexafluorophosphate, using triphenyl phosphite as the condensing agent under mild reaction conditions. The highly hydrophobic ASSP was transformed into a water soluble hydrophilic micelle by coupling ASSP with polyethylene glycol (PEG using the same ionic liquid and 1,1-carbonyl diimidazole as the condensing agent without harsh conditions. The polycationic ASSP-PEG was then used to condense calf thymus and plasmid deoxyribonuclceic acids (DNAs in Tris-HCl buffer (pH 7.4 to get a series of block ionomer complexes with various charge ratios. The physicochemical properties of the copolymer micelle and the DNA polyplexes were studied using fourier transform-infrared (FTIR, nuclear magnetic resonance (NMR and circular dichroism (CD spectroscopy, matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, differential scanning calorimetry (DSC, transmission electron microscopy (TEM and particle size measurements. It was observed that the DNA was condensed to compact particles by its interaction with the copolymer. Since DNA condensation to nano/micrometer sized particles is essential for gene delivery, our results indicate a potential use of the copolymer for gene delivery applications.

  16. Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

    Directory of Open Access Journals (Sweden)

    Venkateswara Reddy Gogulamudi

    2015-10-01

    Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

  17. Static and dynamic investigations of poly(aspartic acid) and Pluronic F127 complex prepared by self-assembling in aqueous solution

    Science.gov (United States)

    Nita, Loredana E.; Chiriac, Aurica P.; Bercea, Maria; Nistor, Manuela T.

    2015-12-01

    The present investigation is focused on evaluation of self-assembling ability in aqueous solutions of two water soluble polymers: poly(aspartic acid) (PAS) and Pluronic F127 (PL). The intermolecular complexes, realized between polyacid and neutral copolymer surfactant in different ratios, have been studied by combining various characterization techniques as rheology, DLS, spectroscopy, microscopy, chemical imaging, and zeta potential determination, measurements performed in static and/or dynamic conditions. In static conditions, when the equilibrium state between PAS/PL polymeric pair was reached, and depending on the polymers mixture composition, and of experimental rheological conditions, positive or negative deviations from the additive rule are registered. Conformational changes of the macromolecular chains and correspondingly physical interactions are generated between PL and PAS for self-assembly and the formation of interpolymer complex as suprastructure with micellar configuration. The phenomenon was better evidenced in case of 1/1 wt ratio between the two polymers. In dynamic conditions of determination, during "in situ" evaluation of the hydrodynamic diameter, zeta potential and conductivity, when the equilibrium state is not reached and as result either the intermolecular bonds are not achieved, the self-assembling process is not so obvious evidenced.

  18. Synthesis and Crystal Structure of a New Ternary Complex of Nickel(Ⅱ) with L-Aspartic Acid and 1,10-Phenanthroline

    Institute of Scientific and Technical Information of China (English)

    马录芳; 梁福沛; 覃海错; 胡瑞祥; 张漫波

    2004-01-01

    A new ternary complex of nickel(Ⅱ) with L-aspartate (L-Asp) and 1, 10-phenanthroline(Phen), [Ni3(L-Asp)(Phen)5(H2O)3](ClO4)4.4.75H2O, has been synthesized in a mixed solvent of H2OC2H5OH at the pH value of 3. It crystallizes in triclinic, space group P-1 with a =11.861(2), b = 18.384(4), c = 19.746(3)(°A), α = 107.68(1), β = 105.94(1), γ= 103.41(2)°, V =3703.6(11) (°A)3, Mr = 1745.66, Z = 2, Dc =1.565 g/cm3, u = 0.989 mm-1, F(000) = 1791, R = 0.0643and wR = 0.1611. The complex contains two cations, binuclear [Ni2(L-Asp)(Phen)3(H2O)]2+ and mononu- clear [Ni(Phen)2(H2O)2]2+. All the nickel ions in the complex are six-coordinated with a distorted octahedral geometry, but the coordination environments for them are different. There exist hydrogen bonds and π-π stacking interactions in the complex.

  19. Exploring details about structure requirements based on novel CGRP receptor antagonists urethanamide, aspartate, succinate and pyridine derivatives by in silico methods

    Science.gov (United States)

    Li, Yan; He, Haoran; Wang, Jinghui; Han, Chunxiao; Feng, Jiaqi; Zhang, Shuwei; Yang, Ling

    2014-09-01

    The migraine never fails to afflict individuals in the world that knows no lack of such cases. CGRP (calcitonin gene-related peptide) is found closely related to migraine and olcegepant (BIBN4096) is effective in alleviating the pain. In our work, the combination of ligand- and receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies along with molecular docking was applied to provide us insights about how urethanamide, pyridine and aspartate and succinate derivatives (novel CGRP receptor antagonists) play a part in inhibiting the activity of CGRP receptor. The optimal CoMSIA model shows the Q2 of 0.505, R2ncv of 0.992 and its accurate predictive ability was confirmed by checking out an independent test set which gave R2pred value of 0.885. Besides, the 3D contour maps help us identify how different groups affect the antagonist activity while connecting to some key positions. In addition, the docking analysis shows the binding site emerging as the distorted “V” shape and including two binding pockets: one of them is hydrophobic, fixing the structural part 3 of compound 80, the other anchors the part 1 of compound 80. The docking analysis also shows the interaction mechanism between compound 80 and CGRP receptor, similar to the interaction between olcegepant and CGRP receptor. The findings derived from this work reveal the mechanism of related antagonists and facilitate the future rational design of novel antagonists with higher potency.

  20. Accumulation of radioactivity in rat brain and peripheral tissues including salivary gland after intravenous administration of {sup 14}C-D-aspartic acid

    Energy Technology Data Exchange (ETDEWEB)

    Imai, Kazuhiro; Fukushima, Takeshi; Santa, Tomofumi; Homma, Hiroshi [Tokyo Univ. (Japan). Faculty of Pharmaceutical Sciences; Sugihara, Juko; Kodama, Hirohiko; Yoshikawa, Masayoshi

    1997-03-01

    After the intravenous administration of {sup 14}C-D-aspartic acid (Asp) into Sprague-Dawley rats (male, 7-week-old), the distribution and elimination of radioactivity was investigated by the whole body autoradiography. High radioactivities were detected in pineal gland, pituitary gland and salivary gland at 30 min after administration. The other tissues detected were liver, lung, adrenal gland, pancreas and spleen where D-Asp was reported to occur naturally. After 24 hr, the radioactivities were still detected at high levels in the pineal, pituitary and salivary glands. The data suggested the natural occurrence of D-Asp in salivary gland. After careful examination utilizing fluorescent derivatization and chiral separation by high-performance liquid chromatography, the presence of D-Asp was, for the first time, demonstrated in salivary gland in situ, the concentration of which was 7.85 {+-} 1.0 nmol/g. The administration of {sup 14}C-L-Asp was also carried out. The data suggested that D-Asp in the circulating blood is one of the sources of the tissue D-Asp. (author)

  1. Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

    Directory of Open Access Journals (Sweden)

    Cui Songqin

    2009-12-01

    Full Text Available Abstract Background Experimental and clinical studies showed that intraoperative infusionof remifentanil has been associated with postoperative hyperalgesia. Previous reports suggested that spinal N-methyl-D-aspartate (NMDA receptors may contribute to the development and maintenance of opioid-induced hyperalgesia. In the present study, we used a rat model of postoperative pain to investigate the role of tyrosine phosphorylation of NMDA receptor 2B (NR2B subunit in spinal cord in the postoperative hyperalgesia induced by remifentanil and the intervention of pretreatment with ketamine. Results Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous significantly enhanced mechanical allodynia and thermal hyperalgesia induced by the plantar incision during the postoperative period (each lasting between 2 h and 48 h, which was attenuated by pretreatment with ketamine (10 mg/kg, subcutaneous. Correlated with the pain behavior changes, immunocytochemical and western blotting experiments in our study revealed that there was a marked increase in NR2B phosphorylation at Tyr1472 in the superficial dorsal horn after intraoperative infusion of remifentanil, which was attenuated by pretreatment with ketamine. Conclusions This study provides direct evidence that tyrosine phosphorylation of the NR2B at Tyr1472 in spinal dosal horn contributes to postoperative hyperalgesia induced by remifentanil and supports the potential therapeutic value of ketamine for improving postoperative hyperalgesia induced by remifentanil.

  2. Spinal Tolerance and Dependence: Some Observations on the Role of Spinal N-Methyl-D-Aspartate Receptors and Phosphorylation in the Loss of Opioid Analgesic Responses

    Directory of Open Access Journals (Sweden)

    Tony L Yaksh

    2000-01-01

    Full Text Available The continuous delivery of opiates can lead to a reduction in analgesic effects. In humans, as in other animals, some component of this change in sensitivity seems likely to have a strong pharmacodynamic component. Such loss of effect, deemed to be tolerance in the present article, can be readily demonstrated in animals with repeated bolus and continuous intrathecal infusion of mu and delta opioids and alpha-2 adrenergic agonists. Research has shown that this loss of effect can be diminished by concurrent treatment with N-methyl-D-aspartate (NMDA receptor antagonists and by the suppression of the activity of spinal protein kinase C (PKC. This suggests in part the probable role of PKC-mediated phosphorylation in the right shift in the dose-effect curves observed with continuous opiate or adrenergic exposure. Importantly, this right shift is seen to occur in parallel with an increase in the phosphorylating activity in the dorsal horn and in the expression of several PKC isozymes. The target of this phosphorylation is not certain. Phosphorylation of the NMDA receptor enhances its functionality, while phosphorylation of the opioid receptor or associated channels seems to diminish their activity or to enhance internalization. While the focus is on several specific components, the accumulating data emphasize the biological complexity of these changes in spinal drug reactivity.

  3. Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD

    Science.gov (United States)

    Aly, Khaled A; Beebe, Emily T; Chan, Chi H; Goren, Michael A; Sepúlveda, Carolina; Makino, Shin-ichi; Fox, Brian G; Forest, Katrina T

    2013-01-01

    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence. PMID:23255525

  4. Biphasic Insulin Aspart 30/70: Pharmacokinetics and Pharmacodynamics Compared With Once-Daily Biphasic Human Insulin and Basal-Bolus Therapy

    Science.gov (United States)

    Heise, Tim; Heinemann, Lutz; Hövelmann, Ulrike; Brauns, Bianca; Nosek, Leszek; Haahr, Hanne L.; Olsen, Klaus J.

    2009-01-01

    OBJECTIVE Pharmacological profiles of biphasic insulin aspart 30/70 (BIAsp 30) once daily (OD), twice daily (b.i.d.), and three times daily (t.i.d.) were compared with other insulin regimens in two crossover glucose clamp studies of insulin-treated type 2 diabetic patients. RESEARCH DESIGNS AND METHODS Study 1 consisted of BIAsp 30 OD, b.i.d., and t.i.d. versus biphasic human insulin 30/70 (BHI 30), OD (n = 24). Study 2 examined BIAsp 30 t.i.d. versus basal-bolus therapy (insulin glargine OD plus insulin glulisine t.i.d.) (n = 24). Pharmacokinetics/pharmacodynamics (PK/PD) were investigated over 24 h. RESULTS Study 1: PK and PD were markedly different between BIAsp 30 OD and BHI 30 OD: the maximum insulin concentration and glucose infusion rate (GIR) were higher for BIAsp 30; time to maximum metabolism was 1.7 h sooner for BIAsp 30. Study 2: both regimens showed three distinct prandial-related GIR peaks. GIR 24-h area under the curve for BIAsp t.i.d. was higher than for basal-bolus therapy: 2,585.2 vs. 2,289.2 mg/kg. CONCLUSIONS BIAsp had pharmacological advantages over BHI. BIAsp t.i.d. had a similar PD profile to basal-bolus therapy. PMID:19487640

  5. N-methyl-D-aspartate (NMDA) and the regulation of mitogen-activated protein kinase (MAPK) signaling pathways: a revolving neurochemical axis for therapeutic intervention?

    Science.gov (United States)

    Haddad, John J

    2005-11-01

    Excitatory synaptic transmission in the central nervous system (CNS) is mediated by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and non-NMDA (AMPA and KA) receptors. Extracellular signals control diverse neuronal functions and are responsible for mediating activity-dependent changes in synaptic strength and neuronal survival. Influx of extracellular calcium ([Ca(2+)](e)) through the NMDA receptor (NMDAR) is required for neuronal activity to change the strength of many synapses. At the molecular level, the NMDAR interacts with signaling modules, which, like the mitogen-activated protein kinase (MAPK) superfamily, transduce excitatory signals across neurons. Recent burgeoning evidence points to the fact that MAPKs play a crucial role in regulating the neurochemistry of NMDARs, their physiologic and biochemical/biophysical properties, and their potential role in pathophysiology. It is the purpose of this review to discuss: (i) the MAPKs and their role in a plethora of cellular functions; (ii) the role of MAPKs in regulating the biochemistry and physiology of NMDA receptors; (iii) the kinetics of MAPK-NMDA interactions and their biologic and neurochemical properties; (iv) how cellular signaling pathways, related cofactors and intracellular conditions affect NMDA-MAPK interactions and (v) the role of NMDA-MAPK pathways in pathophysiology and the evolution of disease conditions. Given the versatility of the NMDA-MAPK interactions, the NMDA-MAPK axis will likely form a neurochemical target for therapeutic interventions.

  6. SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein.

    Science.gov (United States)

    Sakinc, Türkan; Kleine, Britta; Gatermann, Sören G

    2006-08-01

    A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

  7. Aspartate aminotransferase-lymphocyte ratio index and systemic immune-inflammation index predict overall survival in HBV-related hepatocellular carcinoma patients after transcatheter arterial chemoembolization.

    Science.gov (United States)

    Yang, Zongguo; Zhang, Jianliang; Lu, Yunfei; Xu, Qingnian; Tang, Bozong; Wang, Qiang; Zhang, Wensi; Chen, Shishi; Lu, Lingqing; Chen, Xiaorong

    2015-12-15

    It has been suggested that lymphocytes play central roles in host antitumor immune responses and control cancer outcome. We reviewed the clinical parameters of 189 hepatocellular carcinoma (HCC) patients and investigated the prognostic significance of lymphocyte-related scores in HCC patients following transcatheter arterial chemoembolization (TACE). Survival analysis revealed that an elevated aspartate aminotransferase-lymphocyte ratio index (ALRI) > 57 and a systemic immune-inflammation index (SII) > 300 were negatively associated with overall survival in HBV-related HCC (HR = 2.181, P = 0.003 and HR = 2.453, P = 0.003; respectively). Spearman chi-square analysis showed that ALRI had a specificity of 82.4% and that SII index had a sensitivity of 71.9% for HCC overall survival. ALRI and SII had negative predictive values of 74.6% and 80%, respectively for HCC overall survival. Additionally, Barcelona Clinic Liver Cancer (BCLC) stage C patients had significantly higher ALRI and SII scores (both P SII scores (P SII should be used as negative predictive factors for overall survival in HBV-related HCC in clinical practice.

  8. Prediction of the Risk of Hepatocellular Carcinoma in Chronic Hepatitis C Patients after Sustained Virological Response by Aspartate Aminotransferase to Platelet Ratio Index

    Science.gov (United States)

    Lee, Keol; Sinn, Dong Hyun; Gwak, Geum-Youn; Cho, Hyun Chin; Jung, Sin-Ho; Paik, Yong-Han; Choi, Moon Seok; Lee, Joon Hyeok; Koh, Kwang Cheol; Paik, Seung Woon

    2016-01-01

    Background/Aims Following sustained virological response (SVR) for chronic hepatitis C (CHC) infection, patients with advanced fibrosis require regular monitoring for hepatocellular carcinoma (HCC). The aspartate aminotransferase to platelet ratio index (APRI) is a simple noninvasive surrogate marker known to reflect fibrosis. Methods We retrospectively analyzed 598 patients who achieved SVR with interferon-based therapy for CHC. Results Over a median of 5.1 years of follow-up, there were eight patients diagnosed with HCC and a 5-year cumulative incidence rate of 1.3%. The median pretreatment APRI was 0.83, which decreased to 0.29 after achieving SVR (p<0.001). Both the pre- and posttreatment indices were associated with HCC development. The 5-year cumulative HCC incidence rates were 0% and 2.8% for patients with pretreatment APRI <1.0 and ≥1.0, respectively (p=0.001) and 0.8% and 12.8% for patients with posttreatment APRI <1.0 and ≥1.0, respectively (p<0.001). Pretreatment APRI at a cutoff of 1.0 had a 100% negative predictive value until 10 years after SVR. Conclusions HCC development was observed among CHC patients who achieved SVR. The pre- and post-treatment APRI could stratify HCC risk, indicating that the APRI could be a useful marker to classify HCC risk in CHC patients who achieved SVR. However, given the small number of HCC patients, this finding warrants further validation. PMID:27114418

  9. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

    Science.gov (United States)

    Flahaut, M; Sanglard, D; Monod, M; Bille, J; Rossier, M

    1998-02-01

    Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

  10. Application of L-Aspartic Acid-Capped ZnS:Mn Colloidal Nanocrystals as a Photosensor for the Detection of Copper (II Ions in Aqueous Solution

    Directory of Open Access Journals (Sweden)

    Jungho Heo

    2016-04-01

    Full Text Available Water-dispersible ZnS:Mn nanocrystals (NCs were synthesized by capping the surface with polar L-aspartic acid (Asp molecules. The obtained ZnS:Mn-Asp NC product was optically and physically characterized using the corresponding spectroscopic methods. The ultra violet-visible (UV-VIS absorption spectrum and photoluminescence (PL emission spectrum of the NCs showed broad peaks at 320 and 590 nm, respectively. The average particle size measured from the obtained high resolution-transmission electron microscopy (HR-TEM image was 5.25 nm, which was also in accordance with the Debye-Scherrer calculations using the X-ray diffraction (XRD data. Moreover, the surface charge and degree of aggregation of the ZnS:Mn-Asp NCs were determined by electrophoretic and hydrodynamic light scattering methods, respectively. These results indicated the formation of agglomerates in water with an average size of 19.8 nm, and a negative surface charge (−4.58 mV in water at ambient temperature. The negatively-charged NCs were applied as a photosensor for the detection of specific cations in aqueous solution. Accordingly, the ZnS:Mn-Asp NCs showed an exclusive luminescence quenching upon addition of copper (II cations. The kinetic mechanism study on the luminescence quenching of the NCs by the addition of the Cu2+ ions proposed an energy transfer through the ionic binding between the two oppositely-charged ZnS:Mn-Asp NCs and Cu2+ ions.

  11. Efficacy and safety of biphasic insulin aspart and biphasic insulin lispro mix in patients with type 2 diabetes: A review of the literature

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2016-01-01

    Full Text Available Type 2 diabetes (T2D represents an escalating burden worldwide, particularly in China and India. Compared with Caucasians, Asian people with diabetes have lower body mass index, increased visceral adiposity, and postprandial glucose (PPG/insulin resistance. Since postprandial hyperglycemia contributes significantly to total glycemic burden and is associated with heightened cardiovascular risk, targeting PPG early in T2D is paramount. Premixed insulin regimens are widely used in Asia due to their convenience and effectiveness. Data from randomized controlled trials and observational studies comparing efficacy and safety of biphasic insulin aspart 30 (BIAsp 30 with biphasic insulin lispro mix (LM 25/50 and versus other insulin therapies or oral antidiabetic drugs (OADs in T2D demonstrated that BIAsp 30 and LM 25/50 were associated with similar or greater improvements in glycemic control versus comparator regimens, such as basal–bolus insulin, in insulin-naÏve, and prior insulin users. Studies directly comparing BIAsp 30 and LM 25 provided conflicting glycemic control results. Safety data generally showed increased hypoglycemia and weight gain with premixed insulins versus basal–bolus insulin or OADs. However, large observational trials documented improvements in glycated hemoglobin, PPG, and hypoglycemia with BIAsp 30 in multi-ethnic patient populations. In summary, this literature review demonstrates that premixed insulin regimens are an appropriate and effective treatment choice in T2D.

  12. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  13. Ectopic expression of a grape aspartic protease gene, AP13, in Arabidopsis thaliana improves resistance to powdery mildew but increases susceptibility to Botrytis cinerea.

    Science.gov (United States)

    Guo, Rongrong; Tu, Mingxing; Wang, Xianhang; Zhao, Jiao; Wan, Ran; Li, Zhi; Wang, Yuejin; Wang, Xiping

    2016-07-01

    The grape aspartic protease gene, AP13 was previously reported to be responsive, in Chinese wild Vitis quinquangularis cv. 'Shang-24', to infection by Erysiphe necator, the causal agent of powdery mildew disease, as well as to treatment with salicylic acid in V. labrusca×V. vinifera cv. 'Kyoho'. In the current study, we evaluated the expression levels of AP13 in 'Shang-24' in response to salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET) treatments, as well as to infection by the necrotrophic fungus, Botrytis cinerea, and the transcript levels of VqAP13 decreased after B. cinerea infection and MeJA treatment, but increased following ET and SA treatments. Transgenic Arabidopsis thaliana lines over-expressing VqAP13 under the control of a constitutive promoter showed enhanced resistance to powdery mildew and to the bacterium Pseudomonas syringae pv. tomato DC3000, and accumulated more callose than wild type plants, while the resistance of transgenic A. thaliana lines to B. cinerea inoculation was reduced. In addition, the expression profiles of various disease resistance- related genes in the transgenic A. thaliana lines following infection by different pathogens were compared to the equivalent profiles in the wild type plants. The results suggest that VqAP13 action promotes the SA dependent signal transduction pathway, but suppresses the JA signal transduction pathway. PMID:27181943

  14. Effect of Xingnaojing Injection(醒脑静注射液)on Hippocampal N—methyl—D—aspartic Acid Receptors of Focal Cerebral Ischemia in Rats

    Institute of Scientific and Technical Information of China (English)

    SHENSi-yu; CAIDing-fang; 等

    2003-01-01

    Objective:To observe and elucidate the neuroprotective effect of Xingnaojing(XNJ) injec-tion on hippocampal N-methyl-D-aspartic acid(NMDA) receptors of focal cerebral ischemia in rats.Meth-ods:Cerebral ischemia was established by occluding the middle cerebral artery with an intraluminal suture technique in rats.Neurological deficit score,infarct volume and quantity of NMDA receptors were esti-mated in all groups and compared.Results:After being treated with XNJ,the score decreased in the initial 6 hours and infarct volume decreased in 24 hours.And within 24 hours,the quantity of NMDA receptors obviously decreased compared with the model group(P<0.01).It indicated that XNJ could ameliorate neurological behavior of middle cerebral artery occlusion rats and down-regulate the expression of hipp-ocampal NMDA receptors.Conclusion:The neuroprotective effect of XNJ on focal cerebral ischemia is possibly related to down-regulating the expression of NMDA receptors in rats.

  15. Surface aggregation of urinary proteins and aspartic acid-rich peptides on the faces of calcium oxalate monohydrate investigated by in situ force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, M L; Qiu, S R; Hoyer, J R; Casey, W H; Nancollas, G H; De Yoreo, J J

    2008-05-28

    The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin (OPN), and the 27-residue synthetic peptides (DDDS){sub 6}DDD and (DDDG){sub 6}DDD [where D = aspartic acid and X = S (serine) or G (glycine)] was investigated via in situ atomic force microscopy (AFM). The results show that these three growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition or increase of the step speeds (with respect to the impurity-free system) depending on a range of factors that include peptide or protein concentration, supersaturation and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (-101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we argue for a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at crystal surface.

  16. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  17. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  18. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  19. Clinical presentations of pediatric anti-N-methyl-D-aspartate receptor encephalitis%抗N-甲基-D-天门冬氨酸受体脑炎的临床表现

    Institute of Scientific and Technical Information of China (English)

    姜玉武; 季涛云

    2014-01-01

    抗N-甲基-D-天门冬氨酸受体(N-methyl-D-aspartate receptor, NMDAR)脑炎是儿科最常见的一种具有独特临床表现的免疫性脑炎。该文对典型和不典型抗NMDAR脑炎的临床表现及儿童抗NMDAR脑炎临床表现的独特之处等进行了综述,以提高儿科医生对此症的认识及诊治水平。%Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is one of the most common autoimmune encephalitis in children with characterized clinical features. Here we review clinical presentations of typical and atypical anti-NMDAR encephalitis and characteristics of clinical presentations of pediatric anti-NMDAR encephalitis.

  20. Contrôle de la vitesse de germination chez le maïs (Zea mays) : étude de la voie de biosynthèse des acides aminés issus de l'aspartate et recherche de QTLs

    OpenAIRE

    Anzala, Fabiola,

    2006-01-01

    The aspartate pathway was studied through the expression of the genes ask1, ask2 akh1 and akh2 coding for aspartate kinase by qt-RT-PCR, the amino acids content by HPLC and the degradation of asp-15N in the derived amino acids followed by labelling of 15N. It appears that the aspartate pathway differs between two (slow and fast) genotypes of maize: slow germination would be associated with an inhibiting effect of the accumulation of lysine or to limited contents of threonine and methionine. T...

  1. 门冬氨酸鸟氨酸的肝病临床应用与研究进展%Clinical application and pharmacological effect of L-ornithine-L-aspartate in liver diseases

    Institute of Scientific and Technical Information of China (English)

    成军

    2015-01-01

    门冬氨酸鸟氨酸作为一种抗炎保肝药物广泛用于各种肝脏疾病的治疗,如肝性脑病、药物性肝损、脂肪肝、慢性肝炎等。除了三羧酸循环和鸟氨酸循环外,最新的研究显示,门冬氨酸鸟氨酸的抗炎作用可能与N-甲基-D-门冬氨酸(NMDA)受体有关。门冬氨酸鸟氨酸能够促进肝细胞修复,减轻肝脏炎性反应,降低肝细胞损害,临床疗效确切,而新的作用机制也具有进一步的研究价值。%L-ornithine-L-aspartate has been widely used as an anti-inflammatory and hepatoprotective drug in clinic, e.g. hepatic encephalopathy, drug-induced liver damage, fatty liver, chronic hepatitis, etc. Beside krebs cycle and ornithine cycle, recent research showed that the anti-inflammatory effect of L-ornithine-L-aspartate was associated with N-methyl-D-aspartate (NMDA) receptor. L-ornithine-L-aspartate could promote liver cell repair, decrease the liver inflammatory reaction, reduce the liver cell damage. The clinical curative effect is definite, the novel mechanism has also potential research value.

  2. Solid-state conformation of copolymers of ß-benzyl-L-aspartate with L-alanine, L-leucine, L-valine, γ-benzyl-L-glutamate, or ε-carbobenzoxy-L-lysine

    NARCIS (Netherlands)

    Sederel, Willem L.; Bantjes, Adriaan; Feijen, Jan; Anderson, James M.

    1980-01-01

    The solid-state conformation of copolymers of ß-benzyl-L-aspartate [L-Asp(OBzl)] with L-leucine (L-Leu), L-alanine (L-Ala), L-valine (L-Val), γ-benzyl-L-glutamate [L-Glu(OBzl)], or ε-carbobenzoxy-L-lysine (Cbz-L-Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in

  3. SERUM ACTIVITIES OF ASPARTATE AMINOTRANSFERASE, CREATINE KINASE AND LACTATE DEHYDROGENASE IN HORSES WITH COLIC ATIVIDADE SÉRICA DAS ENZIMAS ASPARTATO AMINOTRANSFERASE, CREATINA QUINASE E LACTATO DESIDROGENASE EM EQÜINOS COM CÓLICA

    OpenAIRE

    Aureo Evangelista Santana; Paula Alessandra Di Filippo

    2008-01-01

    Seventy equines distributed in two experimental groups were used, G1 (20 healthy equines), and G2 (50 equines with colic). Blood samples were obtained by jugular vein puncture in ten different moments. The variables aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) were determined by spectrophotometric assay using specific reagents. The average values presented by the animals of the G2 for variables CK, AST, and LDH were higher (P<0.05) than the values...

  4. Demonstration on Fertilizer Effect of Poly Aspartic Acid on Rice%聚天冬氨酸增效肥在水稻上的应用效果示范

    Institute of Scientific and Technical Information of China (English)

    李梅; 沈文忠; 左其东; 张绪美

    2015-01-01

    Objective] The aim was to provide references for poly aspartic acid applied on rice. [Method] The fertilizer effect of poly aspartic acid on rice was studied, by conducting test. [Result] The results showed that rice plant height increased 2.67 cm compared with conventional fertilization way, and increased 2.97%, by applying poly aspartic acid on rice. The effective panicles increased 25500 ears, and increased the amplitude of 0.85%. Empty abortive rate decreased by 7.26%. Rice yield increased significantly with increase of 635.7 kg/hm2, with the production rate of 8.11%. [Conclusion] Application of poly aspartic acid could obtain obvious increase production effect in the process of rice production.%[目的]为聚天冬氨酸增效肥在水稻上的应用提供技术支撑。[方法]针对聚天冬氨酸增效肥在水稻上应用的实际效果进行试验。[结果]结果表明,与常规施肥方式相比,施用了聚天冬氨酸增效肥的水稻株高增加了2.67 cm,增幅为2.97%;有效穗数增加2.55万穗,增幅为0.85%;空秕率减少了7.26%;水稻产量明显增加,增产635.7 kg/hm2,增产率为8.11%。[结论]水稻生产过程中施用聚天冬氨酸增效肥可取得明显增产增收效果。

  5. Ethanol withdrawal is required to produce persisting N-methyl-D-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure

    Science.gov (United States)

    Reynolds, Anna R.; Berry, B. Jennifer N.; Sharrett-Field, Lynda; Prendergast, Mark A.

    2015-01-01

    Chronic intermittent ethanol consumption is associated with neurodegeneration and cognitive deficits in preclinical laboratory animals and in the clinical population. While previous work suggests a role for neuroadaptations in the N-methyl-D-aspartate (NMDA) receptor in the development of ethanol dependence and manifestation of withdrawal, the relative roles of ethanol exposure and ethanol withdrawal in producing these effects have not been fully characterized. To examine underlying cytotoxic mechanisms associated with CIE exposure, organotypic hippocampal slices were exposed to 1–3 cycles of ethanol (50 mM) in cell culture medium for 5 days, followed by 24-hours of ethanol withdrawal in which a portion of slices were exposed to competitive NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 40 µM). Cytotoxicity was assessed using immunohistochemical labeling of neuron specific nuclear protein (NeuN; Fox-3), a marker of mature neurons, and thionine (2%) staining of Nissl bodies. Multiple cycles of CIE produced neurotoxicity, as reflected in persisting losses of neuron NeuN immunoreactivity and thionine staining in each of the primary cell layers of the hippocampal formation. Hippocampi aged in vitro were significantly more sensitive to the toxic effects of multiple CIEs than were non-aged hippocampi. This effect was not demonstrated in slices exposed to continuous ethanol, in the absence of withdrawal, or to a single exposure/withdrawal regimen. Exposure to APV significantly attenuated the cytotoxicity observed in the primary cell layers of the hippocampus. The present findings suggest that ethanol withdrawal is required to produce NMDA receptor-dependent hippocampal cytotoxicity, particularly in the aging hippocampus in vitro. PMID:25746220

  6. Pathologically activated neuroprotection via uncompetitive blockade of N-methyl-D-aspartate receptors with fast off-rate by novel multifunctional dimer bis(propyl)-cognitin.

    Science.gov (United States)

    Luo, Jialie; Li, Wenming; Zhao, Yuming; Fu, Hongjun; Ma, Dik-Lung; Tang, Jing; Li, Chaoying; Peoples, Robert W; Li, Fushun; Wang, Qinwen; Huang, Pingbo; Xia, Jun; Pang, Yuanping; Han, Yifan

    2010-06-25

    Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation. PMID:20404346

  7. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    Science.gov (United States)

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated. PMID:22886112

  8. Prion protein is a key determinant of alcohol sensitivity through the modulation of N-methyl-D-aspartate receptor (NMDAR activity.

    Directory of Open Access Journals (Sweden)

    Agnès Petit-Paitel

    Full Text Available The prion protein (PrP is absolutely required for the development of prion diseases; nevertheless, its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches in transgenic mouse models, we provide strong evidence for a crucial role of PrP in alcohol sensitivity. Indeed, PrP knock out (PrP(-/- mice presented a greater sensitivity to the sedative effects of EtOH compared to wild-type (wt control mice. Conversely, compared to wt mice, those over-expressing mouse, human or hamster PrP genes presented a relative insensitivity to ethanol-induced sedation. An acute tolerance (i.e. reversion to ethanol inhibition of N-methyl-D-aspartate (NMDA receptor-mediated excitatory post-synaptic potentials in hippocampal slices developed slower in PrP(-/- mice than in wt mice. We show that PrP is required to induce acute tolerance to ethanol by activating a Src-protein tyrosine kinase-dependent intracellular signaling pathway. In an attempt to decipher the molecular mechanisms underlying PrP-dependent ethanol effect, we looked for changes in lipid raft features in hippocampus of ethanol-treated wt mice compared to PrP(-/- mice. Ethanol induced rapid and transient changes of buoyancy of lipid raft-associated proteins in hippocampus of wt but not PrP(-/- mice suggesting a possible mechanistic link for PrP-dependent signal transduction. Together, our results reveal a hitherto unknown physiological role of PrP on the regulation of NMDAR activity and highlight its crucial role in synaptic functions.

  9. Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

    Directory of Open Access Journals (Sweden)

    V. Baptista

    2005-01-01

    Full Text Available The nucleus tractus solitarius (NTS plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV. The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

  10. Computer simulations of the interaction of human immunodeficiency virus (HIV) aspartic protease with spherical gold nanoparticles: implications in acquired immunodeficiency syndrome (AIDS)

    Science.gov (United States)

    Whiteley, Chris G.; Lee, Duu-Jong

    2016-09-01

    The interaction of gold nanoparticles (AuNP) with human immune-deficiency virus aspartic protease (HIVPR) is modelled using a regime of molecular dynamics simulations. The simulations of the ‘docking’, first as a rigid-body complex, and eventually through flexible-fit analysis, creates 36 different complexes from four initial orientations of the nanoparticle strategically positioned around the surface of the enzyme. The structural deviations of the enzymes from the initial x-ray crystal structure during each docking simulation are assessed by comparative analysis of secondary structural elements, root mean square deviations, B-factors, interactive bonding energies, dihedral angles, radius of gyration (R g), circular dichroism (CD), volume occupied by C α , electrostatic potentials, solvation energies and hydrophobicities. Normalisation of the data narrows the selection from the initial 36 to one ‘final’ probable structure. It is concluded that, after computer simulations on each of the 36 initial complexes incorporating the 12 different biophysical techniques, the top five complexes are the same no matter which technique is explored. The significance of the present work is an expansion of an earlier study on the molecular dynamic simulation for the interaction of HIVPR with silver nanoparticles. This work is supported by experimental evidence since the initial ‘orientation’ of the AgNP with the enzyme is the same as the ‘final’ AuNP-HIVPR complex generated in the present study. The findings will provide insight into the forces of the binding of the HIVPR to AuNP. It is anticipated that the protocol developed in this study will act as a standard process for the interaction of any nanoparticle with any biomedical target.

  11. Substitution of aspartate for glycine 103 of the type II collagen triple helical domain: Identification of the minimal mutation which can produce Kniest dysplasia

    Energy Technology Data Exchange (ETDEWEB)

    Wilkin, D.J.; Rimoin, D.L.; Cohn, D.H. [Cedars-Sinai Medical Center, Los Angeles, CA (United States)] [and others

    1994-09-01

    Kniest dysplasia is an autosomal dominant chondrodysplasia which results from mutations in the gene for type II collagen, COL2A1. Characteristics of the disorder include a short trunk and extremities, mid-face hypoplasia, cleft palate, myopia, retinal detachment, and hearing loss. Recently, deletions of all or part of exon 12 have been identified in individuals with Kniest dysplasia, suggesting that mutations within this region of the protein may primarily result in the Kniest dysplasia phenotype. We used SSCP to analyze an amplified genomic DNA fragment containing exon 12 from 7 individuals with Kniest dysplasia. An abnormality was identified in one patient. DNA sequence analysis demonstrated that the patient was heterozygous for a G to A transition that implied substitution of glycine{sup 103} of the triple helix by aspartate. The mutation was not observed in DNA from either of the proband`s parents. Protein microsequencing demonstrated expression of the abnormal allele in the proband`s cartilage, indicating that the Kniest phenotype results from the presence of abnormal type II collagen molecules in the extracellular matrix. These data demonstrate the minimal mutation which can produce Kniest dysplasia and further support the hypothesis that alteration of a domain which includes the region encoded by exon 12 in the type II collagen protein leads to this disorder. Experiments designed to identify specific effects that mutations in this region have on intermolecular interactions among abnormal type II collagen molecules and other components of the cartilage extracellular matrix may clarify the underlying pathophysiology of Kniest dysplasia.

  12. Pathologically activated neuroprotection via uncompetitive blockade of N-methyl-D-aspartate receptors with fast off-rate by novel multifunctional dimer bis(propyl)-cognitin.

    Science.gov (United States)

    Luo, Jialie; Li, Wenming; Zhao, Yuming; Fu, Hongjun; Ma, Dik-Lung; Tang, Jing; Li, Chaoying; Peoples, Robert W; Li, Fushun; Wang, Qinwen; Huang, Pingbo; Xia, Jun; Pang, Yuanping; Han, Yifan

    2010-06-25

    Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.

  13. Composite poly-L-lactic acid/poly-(α,β)-DL-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration

    International Nuclear Information System (INIS)

    Tissue engineering scaffolds for skin tissue regeneration is an ever expounding area of research, as the products that meet the necessary requirements are far and elite. The nanofibrous poly-L-lactic acid/poly-(α,β)-DL-aspartic acid/Collagen (PLLA/PAA/Col I and III) scaffolds were fabricated by electrospinning and characterized by SEM, contact angle and FTIR analysis for skin tissue regeneration. The cell-scaffold interactions were analyzed by cell proliferation and their morphology observed in SEM. The results showed that the cell proliferation was significantly increased (p ≤ 0.05) in PLLA/PAA/Col I and III scaffolds compared to PLLA and PLLA/PAA nanofibrous scaffolds. The abundance and accessibility of adipose derived stem cells (ADSCs) may prove to be novel cell therapeutics for dermal tissue regeneration. The differentiation of ADSCs was confirmed using collagen expression and their morphology by CMFDA dye extrusion technique. The current study focuses on the application of PLLA/PAA/Col I and III nanofibrous scaffolds for skin tissue engineering and their potential use as substrate for the culture and differentiation of ADSCs. The objective for inclusion of a novel cell binding moiety like PAA was to replace damaged extracellular matrix and to guide new cells directly into the wound bed with enhanced proliferation and overall organization. This combinatorial epitome of PLLA/PAA/Col I and III nanofibrous scaffold with stem cell therapy to induce the necessary paracrine signalling effect would favour faster regeneration of the damaged skin tissues. - Highlights: ► Differentiation of adipose derived stem cells in the presence of bFGF for wound healing ► Introduction of PAA as ECM mimetic cell binding moiety ► Combination of PLLA/PAA/Col I and III nanofibers and stem cell therapy for skin regeneration.

  14. μ-Opioid and N-methyl-D-aspartate receptors in the amygdala contribute to minocycline-induced potentiation of morphine analgesia in rats.

    Science.gov (United States)

    Ghazvini, Hamed; Rezayof, Ameneh; Ghasemzadeh, Zahra; Zarrindast, Mohammad-Reza

    2015-06-01

    The aim of the present study was to investigate the role of the amygdala in the potentiative effect of minocycline, a semisynthetic tetracycline antibiotic, on morphine analgesia in male Wistar rats. We also examined the involvement of the amygdala μ-opioid and N-methyl-D-aspartate (NMDA) receptors in the minocycline-induced potentiation of morphine analgesia. Intraperitoneal administration of morphine (3-9 mg/kg) induced analgesia in a tail-flick test. Bilateral intra-amygdala injection of minocycline (10-20 μg/rat) enhanced the analgesic response of an ineffective dose of morphine (3 mg/kg). Injection of a higher dose of minocycline into the amygdala also induced analgesia. Moreover, bilateral intra-amygdala injection of naloxone (0.5-1.5 µg/rat) reversed minocycline-induced potentiation of morphine analgesia. Pretreatment of animals with NMDA (0.01-0.1 μg/rat, intra-amygdala) also inhibited the potentiative effect of minocycline on morphine response. Bilateral intra-amygdala injection of the same doses of naloxone or NMDA plus morphine had no effect on the tail-flick latency in the absence of minocycline. It can be concluded that the amygdala has a key role in the potentiative effect of minocycline on morphine analgesia. In addition, amygdala opioidergic and glutamatergic mechanisms may be involved, probably through μ-opioid and NMDA receptors, in the modulation of the minocycline-induced potentiation of morphine analgesia in the tail-flick test. PMID:25563202

  15. Composite poly-L-lactic acid/poly-({alpha},{beta})-DL-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Ravichandran, Rajeswari [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Department of Mechanical Engineering, National University of Singapore, 117576 (Singapore); Venugopal, Jayarama Reddy, E-mail: nnijrv@nus.edu.sg [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Sundarrajan, Subramanian [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Department of Mechanical Engineering, National University of Singapore, 117576 (Singapore); Mukherjee, Shayanti [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Sridhar, Radhakrishnan [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Department of Mechanical Engineering, National University of Singapore, 117576 (Singapore); Ramakrishna, Seeram, E-mail: seeram@nus.edu.sg [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Department of Mechanical Engineering, National University of Singapore, 117576 (Singapore)

    2012-08-01

    Tissue engineering scaffolds for skin tissue regeneration is an ever expounding area of research, as the products that meet the necessary requirements are far and elite. The nanofibrous poly-L-lactic acid/poly-({alpha},{beta})-DL-aspartic acid/Collagen (PLLA/PAA/Col I and III) scaffolds were fabricated by electrospinning and characterized by SEM, contact angle and FTIR analysis for skin tissue regeneration. The cell-scaffold interactions were analyzed by cell proliferation and their morphology observed in SEM. The results showed that the cell proliferation was significantly increased (p {<=} 0.05) in PLLA/PAA/Col I and III scaffolds compared to PLLA and PLLA/PAA nanofibrous scaffolds. The abundance and accessibility of adipose derived stem cells (ADSCs) may prove to be novel cell therapeutics for dermal tissue regeneration. The differentiation of ADSCs was confirmed using collagen expression and their morphology by CMFDA dye extrusion technique. The current study focuses on the application of PLLA/PAA/Col I and III nanofibrous scaffolds for skin tissue engineering and their potential use as substrate for the culture and differentiation of ADSCs. The objective for inclusion of a novel cell binding moiety like PAA was to replace damaged extracellular matrix and to guide new cells directly into the wound bed with enhanced proliferation and overall organization. This combinatorial epitome of PLLA/PAA/Col I and III nanofibrous scaffold with stem cell therapy to induce the necessary paracrine signalling effect would favour faster regeneration of the damaged skin tissues. - Highlights: Black-Right-Pointing-Pointer Differentiation of adipose derived stem cells in the presence of bFGF for wound healing Black-Right-Pointing-Pointer Introduction of PAA as ECM mimetic cell binding moiety Black-Right-Pointing-Pointer Combination of PLLA/PAA/Col I and III nanofibers and stem cell therapy for skin regeneration.

  16. Meta-analysis of insulin aspart versus regular human insulin used in a basal–bolus regimen for the treatment of diabetes mellitus

    Science.gov (United States)

    Heller, Simon; Bode, Bruce; Kozlovski, Plamen; Svendsen, Anne Louise

    2013-01-01

    Background: The objective of the current study was to compare the efficacy of two different insulin formulations, insulin aspart (IAsp) and regular human insulin (RHI), for prandial insulin coverage with neutral protamine Hagedorn (NPH) insulin as basal insulin using a meta-analysis approach. The primary endpoint was change in A1c over time. Secondary endpoints included incidence of hypoglycemia and postprandial glycemic control. Methods Clinical trials (Type 1 and Type 2 diabetes) complying with Good Clinical Practice, and with individual patient data, were included in the meta-analysis. Trials were randomized, consisting of (at least) two treatment arms and had a minimum duration of 12 weeks. Estimates were calculated using fixed-effects and random-effects models. Heterogeneity was assessed for each analysis. The effect of baseline parameters on A1c was analyzed in extended simultaneous models. Results The mean difference in A1c was 0.1% (95% confidence interval [CI] [−0.15; −0.04], P < 0.001) in favor of IAsp. Higher accumulated dose of IAsp, higher age and increased rates of hypoglycemia were associated with improved A1c outcome. Fasting plasma glucose was not significantly different between regimens. Postprandial glucose was significantly lower after treatment with IAsp compared with RHI, but the analysis did present a significant level of heterogeneity (P < 0.001). The overall rate of hypoglycemia was the same with both regimens, but nocturnal hypoglycemia was significantly lower with IAsp. Conclusions A basal–bolus regimen with IAsp as bolus insulin provided minimal, but statistically significant, improvement in overall glycemic control with a lower rate of nocturnal hypoglycemic episodes, compared with a corresponding regimen with bolus RHI. PMID:23586846

  17. Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

    Science.gov (United States)

    Chay, Andrew; Zamparo, Ilaria; Koschinski, Andreas; Zaccolo, Manuela; Blackwell, Kim T

    2016-02-01

    Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

  18. Control of βAR- and N-methyl-D-aspartate (NMDA Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

    Directory of Open Access Journals (Sweden)

    Andrew Chay

    2016-02-01

    Full Text Available Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs, facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs. To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA, and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

  19. The Neuroprotective Effect of Cannabinoid Receptor Agonist (WIN55,212-2 in Paraoxon Induced Neurotoxicity in PC12 Cells and N-methyl-D-aspartate Receptor Interaction

    Directory of Open Access Journals (Sweden)

    Hedayat Sahraei

    2010-01-01

    Full Text Available Objective: Considering that cannabinoids protect neurons against neurodegeneration, inthis study, the neuroprotective effect of WIN55,212-2 in paraoxon induced neurotoxicity inPC12 cells and the role of the N-methyl-D-aspartate (NMDA receptor were evaluated.Materials and Methods: In this study PC12 cells were maintained in Dulbecco's modifiedeagle’s medium (DMEM+F12 culture medium supplemented with 10% fetal bovineserum. The cells were treated with paraoxon (200 μM in the presence or absence ofWIN55,212-2 (0.1 μM, NMDA receptor agonist NMDA (100 μM, cannabinoid receptorantagonist AM251 and NMDA receptor antagonist MK801 (1 μM at 15 minutes intervals.After 48 hours of exposure, cellular viability and protein expression of the CB1 receptorwere evaluated in PC12 cells.Results: Following the exposure of PC12 cells to paraoxon (200 μM, a reduction in cellsurvival and protein level of the CB1 receptor was observed (p<0.01. Treatment of thecells with WIN55,212-2 (0.1 μM and NMDA (100 μM prior to paraoxon exposure significantlyelevated cell survival and protein level of the CB1 receptor (p<0.01. Also, AM251(1μM did not inhibit the cell survival and protein level of the CB1 receptor increase inducedby WIN55,212-2 (p<0.001. However, MK801 (1 μM did inhibit cell survival andprotein expression of the CB1 receptor increase induced by NMDA (p<0.001.Conclusion: The results indicate that WIN55,212-2 and NMDA protect PC12 cellsagainst paraoxon induced toxicity. In addition, the neuroprotective effect of WIN55,212-2and NMDA was cannabinoid receptor-independent and NMDA receptor dependent, respectively.

  20. Effects of ketamine and N-methyl-D-aspartate on fluoxetine-induced antidepressant-related behavior using the forced swimming test.

    Science.gov (United States)

    Owolabi, Rotimi Adegbenga; Akanmu, Moses Atanda; Adeyemi, Oluwole Isaac

    2014-04-30

    This study investigated the effects of ketamine on fluoxetine-induced antidepressant behavior using the forced swimming test (FST) in mice. In order to understand the possible role of N-methyl-d-aspartate (NMDA) neurotransmission in the antidepressant effect of fluoxetine, different groups of mice (n=10) were administered with acute ketamine (3mg/kg, i.p.), acute NMDA (75mg/kg and 150mg/kg, i.p.) and a 21-day chronic ketamine (15mg/kg, i.p./day) were administered prior to the administration of fluoxetine (20mg/kg, i.p.) in the mice. Antidepressant related behavior (immobility score) was measured using the forced swimming test. The results showed that the acute ketamine and fluoxetine alone treatments elicited a significant (pimmobility score compared with saline control. Furthermore, pre-treatment with acute ketamine significantly enhanced by the fluoxetine-induced decrease in immobility score. In contrast, pre-treatment with NMDA (150mg/kg) significantly (pimmobility score. On the other hand, chronic administration of ketamine significantly elicited an increase in immobility score as well as reversed the reduction induced by fluoxetine. Similarly, NMDA administration at both 75mg/kg and 150mg/kg increased immobility score in chronically administered ketamine groups. Furthermore, chronic administration of ketamine, followed by NMDA (75mg/kg) and fluoxetine significantly elevated the immobility score when compared with the group that received NMDA and fluoxetine but not chronically treated with ketamine. It can be suggested) that facilitation of NMDA transmission blocked fluoxetine-induced reduction in immobility score, while down-regulation of NMDA transmission is associated with increase in fluoxetine-induced antidepressant-related behavior in mice. Down-regulation of the NMDA transmission is proposed as an essential component of mechanism of suppression of depression related behaviors by fluoxetine. Modulation of NMDA transmission is suggested to be relevant in

  1. Computer simulations of the interaction of human immunodeficiency virus (HIV) aspartic protease with spherical gold nanoparticles: implications in acquired immunodeficiency syndrome (AIDS).

    Science.gov (United States)

    Whiteley, Chris G; Lee, Duu-Jong

    2016-09-01

    The interaction of gold nanoparticles (AuNP) with human immune-deficiency virus aspartic protease (HIVPR) is modelled using a regime of molecular dynamics simulations. The simulations of the 'docking', first as a rigid-body complex, and eventually through flexible-fit analysis, creates 36 different complexes from four initial orientations of the nanoparticle strategically positioned around the surface of the enzyme. The structural deviations of the enzymes from the initial x-ray crystal structure during each docking simulation are assessed by comparative analysis of secondary structural elements, root mean square deviations, B-factors, interactive bonding energies, dihedral angles, radius of gyration (R g), circular dichroism (CD), volume occupied by C α , electrostatic potentials, solvation energies and hydrophobicities. Normalisation of the data narrows the selection from the initial 36 to one 'final' probable structure. It is concluded that, after computer simulations on each of the 36 initial complexes incorporating the 12 different biophysical techniques, the top five complexes are the same no matter which technique is explored. The significance of the present work is an expansion of an earlier study on the molecular dynamic simulation for the interaction of HIVPR with silver nanoparticles. This work is supported by experimental evidence since the initial 'orientation' of the AgNP with the enzyme is the same as the 'final' AuNP-HIVPR complex generated in the present study. The findings will provide insight into the forces of the binding of the HIVPR to AuNP. It is anticipated that the protocol developed in this study will act as a standard process for the interaction of any nanoparticle with any biomedical target.

  2. N-methyl-D-aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model.

    Science.gov (United States)

    Peng, Wei-Feng; Ding, Jing; Li, Xin; Fan, Fan; Zhang, Qian-Qian; Wang, Xin

    2016-01-01

    Depression is a common comorbidity in patients with epilepsy with unclear mechanisms. This study is to explore the role of glutamate N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. Lithium chloride (Licl)-pilocarpine chronic rat epilepsy model was established and rats were divided into epilepsy with depression (EWD) and epilepsy without depression (EWND) subgroups based on forced swim test. Expression of NMDA receptor NR1, NR2A and NR2B subunits was measured by western blot and immunofluorescence methods. The immobility time (IMT) was significantly greater in Licl-pilocarpine model group than in Control group, which was also greater in EWD group than in EWND group. No differences of spontaneous recurrent seizure (SRS) counts over two weeks and latency were found between EWD and EWND groups. The number of NeuN positive cells was significantly less in Licl-pilocarpine model group than in Control group, but had no difference between EWD and EWND groups. The ratios of phosphorylated NR1 (p-NR1)/NR1 and p-NR2B/NR2B were significantly greater in the hippocampus in EWD group than in EWND group. Moreover, the expression of p-NR1 and p-NR2B in the CA1 subfield of hippocampus were both greater in Licl-pilocarpine model group than Control group. Selective blockage of NR2B subunit with ifenprodil could alleviate depression-like behaviours of Licl-pilocarpine rat epilepsy model. In conclusion, glutamate NMDA receptor NR2B subunit was involved in promoting depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model and might be a target for treating epilepsy-associated depression.

  3. N-methyl-D-Aspartate Receptors Contribute to Complex Spike Signaling in Cerebellar Purkinje Cells: An In vivo Study in Mice.

    Science.gov (United States)

    Liu, Heng; Lan, Yan; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) are post-synaptically expressed at climbing fiber-Purkinje cell (CF-PC) synapses in cerebellar cortex in adult mice and contributed to CF-PC synaptic transmission under in vitro conditions. In this study, we investigated the role of NMDARs at CF-PC synapses during the spontaneous complex spike (CS) activity in cerebellar cortex in urethane-anesthetized mice, by in vivo whole-cell recording technique and pharmacological methods. Under current-clamp conditions, cerebellar surface application of NMDA (50 μM) induced an increase in the CS-evoked pause of simple spike (SS) firing accompanied with a decrease in the SS firing rate. Under voltage-clamp conditions, application of NMDA enhanced the waveform of CS-evoked inward currents, which expressed increases in the area under curve (AUC) and spikelet number of spontaneous CS. NMDA increased the AUC of spontaneous CS in a concentration-dependent manner. The EC50 of NMDA for increasing AUC of spontaneous CS was 33.4 μM. Moreover, NMDA significantly increased the amplitude, half-width and decay time of CS-evoked after-hyperpolarization (AHP) currents. Blockade of NMDARs with D-(-)-2-amino-5-phosphonopentanoic acid (D-APV, 250 μM) decreased the AUC, spikelet number, and amplitude of AHP currents. In addition, the NMDA-induced enhancement of CS activity could not be observed after α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors were blocked. The results indicated that NMDARs of CF-PC synapses contributed to the spontaneous CS activity by enhancing CS-evoked inward currents and AHP currents. PMID:27445699

  4. Subchronic treatment with phencyclidine in adolescence leads to impaired exploratory behavior in adult rats without altering social interaction or N-methyl-D-aspartate receptor binding levels.

    Science.gov (United States)

    Metaxas, A; Willems, R; Kooijman, E J M; Renjaän, V A; Klein, P J; Windhorst, A D; Donck, L Ver; Leysen, J E; Berckel, B N M van

    2014-11-01

    Although both the onset of schizophrenia and human phencyclidine (PCP) abuse typically present within the interval from adolescence to early adulthood, the majority of preclinical research employing the PCP model of schizophrenia has been conducted on neonatal or adult animals. The present study was designed to evaluate the behavioral and neurochemical sequelae of subchronic exposure to PCP in adolescence. Male 35-42-day-old Sprague Dawley rats were subcutaneously administered either saline (10 ml · kg(-1) ) or PCP hydrochloride (10 mg · kg(-1) ) once daily for a period of 14 days (n = 6/group). The animals were allowed to withdraw from treatment for 2 weeks, and their social and exploratory behaviors were subsequently assessed in adulthood by using the social interaction test. To examine the effects of adolescent PCP administration on the regulation of N-methyl-D-aspartate receptors (NMDARs), quantitative autoradiography was performed on brain sections of adult, control and PCP-withdrawn rats by using 20 nM (3) H-MK-801. Prior subchronic exposure to PCP in adolescence had no enduring effects on the reciprocal contact and noncontact social behavior of adult rats. Spontaneous rearing in response to the novel testing arena and time spent investigating its walls and floor were reduced in PCP-withdrawn animals compared with control. The long-term behavioral effects of PCP occurred in the absence of persistent deficits in spontaneous locomotion or self-grooming activity and were not mediated by altered NMDAR density. Our results document differential effects of adolescent PCP administration on the social and exploratory behaviors of adult rats, suggesting that distinct neurobiological mechanisms are involved in mediating these behaviors. PMID:24953757

  5. N-methyl-D-aspartate receptor-mediated glutamate transmission in nucleus accumbens plays a more important role than that in dorsal striatum in cognitive flexibility

    Directory of Open Access Journals (Sweden)

    Xuekun eDing

    2014-09-01

    Full Text Available Cognitive flexibility is a critical ability for adapting to an ever-changing environment in humans and animals. Deficits in cognitive flexibility are observed in most schizophrenia patients. Previous studies reported that the medial prefrontal cortex-to-ventral striatum and orbital frontal cortex-to-dorsal striatum circuits play important roles in extra- and intra-dimensional strategy switching, respectively. However, the precise function of striatal subregions in flexible behaviors is still unclear. N-methyl-D-aspartate receptors (NMDARs are major glutamate receptors in the striatum that receive glutamatergic projections from the frontal cortex. The membrane insertion of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs depends on NMDAR activation and is required in learning and memory processes. In the present study, we measured set-shifting and reversal learning performance in operant chambers in rats and assessed the effects of blocking NMDARs and Ca2+-permeable AMPARs in striatal subregions on behavioral flexibility. The blockade of NMDARs in the nucleus accumbens (NAc core by AP5 impaired set-shifting ability by causing a failure to modify prior learning. The suppression of NMDAR-mediated transmission in the NAc shell induced a deficit in set-shifting by disrupting the learning and maintenance of novel strategies. During reversal learning, infusions of AP5 into the NAc shell and core impaired the ability to learn and maintain new strategies. However, behavioral flexibility was not significantly affected by blocking NMDARs in the dorsal striatum. We also found that the blockade of Ca2+-permeable AMPARs by NASPM in any subregion of the striatum did not affect strategy switching. These findings suggest that NMDAR-mediated glutamate transmission in the NAc contributes more to cognitive execution compared with the dorsal striatum.

  6. Conserved aspartate and lysine residues of RcsB are required for amylovoran biosynthesis, virulence, and DNA binding in Erwinia amylovora.

    Science.gov (United States)

    Ancona, Veronica; Chatnaparat, Tiyakhon; Zhao, Youfu

    2015-08-01

    In Erwinia amylovora, the Rcs phosphorelay system is essential for amylovoran production and virulence. To further understand the role of conserved aspartate residue (D56) in the phosphor receiver (PR) domain and lysine (K180) residue in the function domain of RcsB, amino acid substitutions of RcsB mutant alleles were generated by site-directed mutagenesis and complementation of various rcs mutants were performed. A D56E substitution of RcsB, which mimics the phosphorylation state of RcsB, complemented the rcsB mutant, resulting in increased amylovoran production and gene expression, reduced swarming motility, and restored pathogenicity. In contrast, D56N and K180A or K180Q substitutions of RcsB did not complement the rcsB mutant. Electrophoresis mobility shift assays showed that D56E, but not D56N, K180Q and K180A substitutions of RcsB bound to promoters of amsG and flhD, indicating that both D56 and K180 are required for DNA binding. Interestingly, the RcsBD56E allele could also complement rcsAB, rcsBC and rcsABCD mutants with restored virulence and increased amylovoran production, indicating that RcsB phosphorylation is essential for virulence of E. amylovora. In addition, mutations of T904 and A905, but not phosphorylation mimic mutation of D876 in the PR domain of RcsC, constitutively activate the Rcs system, suggesting that phosphor transfer is required for activating the Rcs system and indicating both A905 and T904 are required for the phosphatase activity of RcsC. Our results demonstrated that RcsB phosphorylation and dephosphorylation, phosphor transfer from RcsC are essential for the function of the Rcs system, and also suggested that constitutive activation of the Rcs system could reduce the fitness of E. amylovora.

  7. Rehabilitation for a child with recalcitrant anti-N-methyl-D-aspartate receptor encephalitis: case report and literature review

    Directory of Open Access Journals (Sweden)

    Guo YH

    2014-11-01

    Full Text Available Yao-Hong Guo,1 Ta-Shen Kuan,1,2 Pei-Chun Hsieh,1 Wei-Chih Lien,1 Chun-Kai Chang,1 Yu-Ching Lin1–3 1Department of Physical Medicine and Rehabilitation, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2Department of Physical Medicine and Rehabilitation, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 3Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan Abstract: Anti-N-methyl-d-aspartate (anti-NMDA receptor encephalitis is a newly recognized, potentially fatal, but treatable autoimmune disease. Good outcome predictors include milder severity of symptoms, no need for intensive care unit admission, early aggressive immunotherapy, and prompt tumor removal. We report a case of a young girl aged 3 years 2 months and diagnosed as recalcitrant anti-NMDA receptor encephalitis without any underlying neoplasm. The patient had initial symptoms of behavioral changes that progressed to generalized choreoathetosis and orofacial dyskinesia, which resulted in 6 months of hospitalization in the pediatric intensive care unit. One year after initial onset of the disease, she had only achieved the developmental age of an infant aged 6–8 months in terms of gross and fine motor skills, but she resumed total independence in activities of daily living after receiving extensive immunotherapy and 28 months of rehabilitation. Our brief review will help clinical practitioners become more familiar with this disease and the unique rehabilitation programs. Keywords: anti-NMDA receptor encephalitis, autoimmune encephalitis, rehabilitation, cognition deficits

  8. Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta-analysis

    Directory of Open Access Journals (Sweden)

    Jin Wenwen

    2012-02-01

    Full Text Available Abstract Background The aspartate aminotransferase-to-platelet ratio index (APRI, a tool with limited expense and widespread availability, is a promising noninvasive alternative to liver biopsy for detecting hepatic fibrosis. The objective of this study was to systematically review the performance of the APRI in predicting significant fibrosis and cirrhosis in hepatitis B-related fibrosis. Methods Areas under summary receiver operating characteristic curves (AUROC, sensitivity and specificity were used to examine the accuracy of the APRI for the diagnosis of hepatitis B-related significant fibrosis and cirrhosis. Heterogeneity was explored using meta-regression. Results Nine studies were included in this meta-analysis (n = 1,798. Prevalence of significant fibrosis and cirrhosis were 53.1% and 13.5%, respectively. The summary AUCs of the APRI for significant fibrosis and cirrhosis were 0.79 and 0.75, respectively. For significant fibrosis, an APRI threshold of 0.5 was 84% sensitive and 41% specific. At the cutoff of 1.5, the summary sensitivity and specificity were 49% and 84%, respectively. For cirrhosis, an APRI threshold of 1.0-1.5 was 54% sensitive and 78% specific. At the cutoff of 2.0, the summary sensitivity and specificity were 28% and 87%, respectively. Meta-regression analysis indicated that the APRI accuracy for both significant fibrosis and cirrhosis was affected by histological classification systems, but not influenced by the interval between Biopsy & APRI or blind biopsy. Conclusion Our meta-analysis suggests that APRI show limited value in identifying hepatitis B-related significant fibrosis and cirrhosis.

  9. 儿童抗N-甲基-D-天冬氨酸受体脑炎%Anti-N-methyl-D-aspartate receptor encephalitis in children

    Institute of Scientific and Technical Information of China (English)

    陈晨

    2013-01-01

    抗N-甲基-D-天冬氨酸受体(N-methyl-D-asparate receptor,NMDAR)脑炎是免疫介导的中枢神经系统综合征,主要临床表现有精神症状、运动障碍、抽搐发作等,常被误诊为病毒性脑炎,与肿瘤尤其卵巢畸胎瘤关系密切;儿童患病率高,抗NMDAR抗体是其较特异的检查;治疗主要为免疫抑制,一线治疗为激素、免疫球蛋白、血浆置换,二线治疗主要包括生物制剂、细胞毒药物;早期诊断及治疗对神经系统的恢复至关重要,总体预后良好.%Anti-N-methyl-D-aspartate receptor encephalitis is an autoimmune syndrome of central nervous system.The main clinical manifestations include psychiatric symptoms,movement disorders and seizures.It is often misdiagnosed as viral encephalitis.Tumors,especially the ovarian teratoma,play an important role in the disease.The prevalence in children is beyond imagination.Anti-NMDAR antibodies are specific diagnostic tests.Immune suppression is the main treatment.The first-line treatments include corticosteroids,intravenous immunoglobulin,and plasmapheresis.The second-line treatments include biological agents and cytotoxic drugs.Early diagnosis and treatment are essential for neurologic recovery.Overall prognosis is good.

  10. Aspartate aminotransferase (AST) blood test

    Science.gov (United States)

    ... Saunders; 2016:chap 73. Read More Acute kidney failure Acute pancreatitis Alanine transaminase (ALT) blood test ALP - blood test Burns Cardiac catheterization Enzyme Heart attack Hemolytic anemia Hepatic Liver cancer - hepatocellular carcinoma Liver disease Mononucleosis Muscular ...

  11. A 2-way cross-over, open-labeled trial to compare efficacy and safety of insulin Aspart and Novolin R delivered with CSII in 21 Chinese diabetic patients

    Institute of Scientific and Technical Information of China (English)

    BI Yu-fang; NING Guang; ZHAO Lie-bin; LI Xiao-ying; WANG Wei-qing; SUN Shou-yue; CHEN Yu-hong; HONG Jie; SU Ting-wei; LIU Jian-min

    2007-01-01

    Background Subcutaneous absorption is accelerated by the monomeric conformation of insulin Aspart, which provides good glycemic control with a lower risk of hypoglycemia and less body weight increase. In the present study we investigated the efficacy and safety of a rapid-acting human insulin analogue (insulin Aspart) delivered with continuous subcutaneous insulin infusion (CSII) into Chinese diabetic patients.Methods Atotal of 21 patients with type 1 or type 2 diabetes were recruited for the 2-way cross-over, open-labeled trial,and then randomized to Group A (n=10, treated with insulin Aspart) or Group B (n=11, treated with Novolin R). Insulin Aspart and Novolin R were administered by CSII. Capillary glucose concentrations were measured at 8 time points,pre-prandial and postprandial, bedtime (10 pm), midnight (2 am) every day during the treatment.Results The average capillary glucose profiles for the day were much better controlled in Group A than in Group B (P<0.01). The blood glucose levels were particularly better controlled in Group A than in Group B at pre-breakfast ((6.72±1.24) mmol/L vs (7.84±1.58) mmol/L, P=0.014), post-breakfast ((8.96±2.41) mmol/L vs (11.70±3.11) mmol/L,P=0.0028), post-supper ((8.15±2.10) mmol/L vs (10.07±2.36) mmol/L, P=0.008), bed time ((7.73±1.72) mmol/L vs (9.39±2.05) mmol/L, P=0.007) and midnight ((6.32±1.16) mmol/L vs (7.48±1.36) mmol/L, P=0.0049). There was no significant difference in the frequency of hypoglycemic episodes between the two groups.Conclusion Insulin Aspart results in better control of blood glucose levels than regular human insulin (Novolin R) in diabetic patients during delivery by CSII.

  12. Initiating therapy or switching to biphasic insulin aspart improves glycaemic control in type 2 diabetes: an Indian experience from the A1chieve study.

    Science.gov (United States)

    Kumar, A; Sharma, S K; Rajput, R; Unnikrishnan, A G

    2013-01-01

    Biphasic insulin aspart 30 (BIAsp 30) has been used in patients for almost a decade; There is a wealth of knowledge from clinical trials to document its efficacy and safety and suggest that BIAsp 30 is an option for initiation and intensification of insulin therapy in patients with type 2 diabetes mellitus (T2DM). The A1chieve was a non-interventional study that explored the safety and effectiveness of initiating or switching to insulin analogues in routine clinical practice in more than 60,000 patients from 28 different countries. In this manuscript, we discuss the findings from the subgroup of the Indian cohort who were treated with BIAsp 30. In a cohort of 15287 who were on BIAsp 30, 12645 (83%) were insulin naive and 2642 (17%) had been on insulin therapy earlier. Glycaemic parameters were high at baseline. Mean (SD) HbA1c was 9.2% (1.3) in the these and was comparable in the insulin naive and insulin experienced groups. After 24 weeks of therapy with BIAsp 30, there were reductions in HbA1c in both the insulin naive group, [-1.8 (1.3)] and insulin experienced group [-1.6 (1.3)]. Fasting plasma glucose (FPG) and postprandial plasma glucose (PPG) levels were also reduced significantly from baseline [-3.4 (2.7) and -4.8 (3.8) mmol/L, respectively, p < 0.05). Overall, hypoglycaemia decreased from 1.33 events/patient years at baseline to 0.19 events/patient years at 24 weeks. There was also an increase in quality of life score as evaluated by EQ-5D questionnaire. Initiating insulin therapy with or switching to BIAsp 30 in patients with poor glycaemic control leads to an improvement in glycaemic profile with no major hypoglycaemia or clinically significant weight gain. Therapy with BIAsp 30 also improves the quality of life in patients with type 2 diabetes.

  13. Distribution of radiolabeled L-glutamate and D-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

    Energy Technology Data Exchange (ETDEWEB)

    Klin, Yael [Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100 (Israel); Zlotnik, Alexander; Boyko, Matthew; Ohayon, Sharon; Shapira, Yoram [The Division of Anesthesiology, Soroka Medical Center and Ben Gurion University of the Negev, Beer-Sheva (Israel); Teichberg, Vivian I., E-mail: Vivian.teichberg@weizmann.ac.il [Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100 (Israel)

    2010-09-03

    Research highlights: {yields} Blood glutamate has a half-life time of 2-3 min. {yields} Blood glutamate is submitted to rapid decarboxylation. {yields} Blood glutamate and its metabolites are mainly absorbed in skeletal muscle and liver. {yields} The skeletal muscle and liver are now targets for potential drugs affording brain neuroprotection. -- Abstract: Excess L-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either L-[1-{sup 14}C] Glutamic acid (L-[1-{sup 14}C] Glu), L-[G-{sup 3}H] Glutamic acid (L-[G-{sup 3}H] Glu) or D-[2,3-{sup 3}H] Aspartic acid (D-[2,3-{sup 3}H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with L-[1-{sup 14}C] Glu and L-[G-{sup 3}H] Glu was faster than that associated with glutamate non-metabolized analog, D-[2,3-{sup 3}H] Asp. L-[1-{sup 14}C] Glu was subjected in blood to a rapid decarboxylation with the loss of {sup 14}CO{sub 2}. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total L-[U-{sup 14}C] Glu or D-[2,3-{sup 3}H] Asp radioactivity capture. L-[U-{sup 14}C] Glu and D-[2,3-{sup 3}H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues

  14. N-methyl-D-aspartate receptor - nitric oxide synthase pathway in the cortex of Nogo-A-deficient rats in relation to brain laterality and schizophrenia

    Directory of Open Access Journals (Sweden)

    Zdena eKristofikova

    2013-08-01

    Full Text Available It has been suggested that Nogo-A, a myelin-associated protein, could play a role in the pathogenesis of schizophrenia and that Nogo-A-deficient rodents could serve as an animal model for schizophrenic symptoms. Since changes in brain laterality are typical of schizophrenia, we investigated whether Nogo-A-deficient rats showed any signs of disturbed asymmetry in cortical N-methyl-D-aspartate (NMDA receptor–nitric oxide synthase (NOS pathway, which is reported as dysfunctional in schizophrenia. In particular, we measured separately in the right and left hemisphere of young and old Nogo-A-deficient male rats the expression of NMDA receptor subunits (NR1, NR2A and NR2B in the frontal cortex and activities of NOS isoforms (neuronal (nNOS, endothelial (eNOS and inducible (iNOS in the parietal cortex. In young controls, we observed right/left asymmetry of iNOS activity and three positive correlations (between NR1 in the left and NR2B laterality, between NR2B in the right and left sides, and between NR1 in the right side and nNOS laterality. In old controls, we found bilateral decreases in NR1, an increase in NR2B in the right side and two changes in correlations in the NR1–nNOS pathway. In young Nogo-A-deficient rats, we observed an increase in iNOS activity in the left hemisphere and two changes in correlations in NR1–nNOS and NR2A–eNOS, compared to young controls. Finally, we revealed in old Nogo-A-deficient animals, bilateral decreases in NR1 and one change in correlation between eNOS–iNOS, compared to old controls. Although some findings from schizophrenic brains did not manifest in Nogo-A-deficient rats (e.g., no alterations in NR2B, others did (e.g., alterations demonstrating accelerated ageing in young but not old animals, those occurring exclusively in the right hemisphere in young and old animals and those suggesting abnormal frontoparietal cortical interactions in young animals.

  15. Expression and distribution of N-methyl-D-aspartate receptor 2A/B in anterior thalamic nuclei and hippocampus of rats

    Institute of Scientific and Technical Information of China (English)

    Kai Fan; Xiaokai Ma; Bin Wang

    2006-01-01

    BACKGROUND: Several immunohistochemical and in situ hybridization studies have demonstrated the localization of various N-methyl-D-aspartate receptors (NMDAR) subunits in certain brain regions of mammals. These studies indicate that NMDAR are widespread in mammal brains, especially in cerebral cortex and hippocam pus.OBJECTIVE: To validate whether or not the NMDAR 2A/B localizes in rat anterior thalamic nuclei (ATN) and hippocampus, and observe its expression and distribution characteristics. DESIGN: Controlled study.SETTING: Department of Anatomy, College of Basic Medicine, Dalian Medical University. MATERIALS: Ten adult Sprague-Dawley rats of either gender, weighing 180-250 g, of clean grade, were provided by the Experimental Animal Center, Dalian Medical University. Rabbit polyclonal antibody to NMDAR 2A/B was the product of Chemicon Company.METHODS: This experiment was carried out in the Department of Anatomy, College of Basic Medicine, Dalian Medical University from September 2005 to May 2006. The anesthetized SD rats were transcardially perfused with 100 mL phosphate buffer solution, then perfused with paraformaldehyde through ascending aorta. After 30 minutes, their brains were harvested and post-fixed in PFA at 4 ℃ overnight. Hippocampal and anterior thalamic nuclear tissues were sliced in the coronal plane at 40 μm. The sections were stained by immunohistochemical ABC method, visualized by DAB, dehydrated routinely, performed transparence and coverslipped. Expression and distribution of NMDAR 2A/B in ATN and hippocampus of rats were observed under an optical microscope.MAIN OUTCOME MEASURES: Distribution and expression of NMDAR 2A/B in ATN and hippocampus of rats, RESULTS: NMDAR 2A/B positive neurons localized in ATN, pyramidal layer of hippocampus and granular layer of dentate gyrus in a compact and orderly pattern, and immunostained intensely. NMDAR 2A/B receptor positive neurons scattered with lower intensity of immunolabeling in other regions of

  16. Optical imaging of head and neck squamous cell carcinoma in vivo using arginine-glycine- aspartic acid peptide conjugated near-infrared quantum dots

    Directory of Open Access Journals (Sweden)

    Huang H

    2013-12-01

    Full Text Available Hao Huang, Yun-Long Bai, Kai Yang, Hong Tang, You-Wei WangDepartment of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of ChinaAbstract: Molecular imaging plays a key role in personalized medicine and tumor diagnosis. Quantum dots with near-infrared emission spectra demonstrate excellent tissue penetration and photostability, and have recently emerged as important tools for in vivo tumor imaging. Integrin αvβ3 has been shown to be highly and specifically expressed in endothelial cells of tumor angiogenic vessels in almost all types of tumors, and specifically binds to the peptide containing arginine-glycine-aspartic acid (RGD. In this study, we conjugated RGD with quantum dots with emission wavelength of 800 nm (QD800 to generate QD800-RGD, and used it via intravenous injection as a probe to image tumors in nude mice bearing head and neck squamous cell carcinoma (HNSCC. Twelve hours after the injection, the mice were still alive and were sacrificed to isolate tumors and ten major organs for ex vivo analysis to localize the probe in these tissues. The results showed that QD800-RGD was specifically targeted to integrin αvβ3 in vitro and in vivo, producing clear tumor fluorescence images after the intravenous injection. The tumor-to-background ratio and size of tumor image were highest within 6 hours of the injection and declined significantly at 9 hours after the injection, but there was still a clearly visible tumor image at 12 hours. The greatest amount of QD800-RGD was found in liver and spleen, followed by tumor and lung, and a weak fluorescence signal was seen in tibia. No detectable signal of QD800-RGD was found in brain, heart, kidney, testis, stomach, or intestine. Our study demonstrated that using integrin αvβ3 as target, it is possible to use intravenously injected QD800-RGD to generate high quality images of HNSCC, and the technique offers great potential

  17. Comparison of thrice daily biphasic human insulin (30/70) versus basal detemir & bolus aspart in patients with poorly controlled type 2 diabetes mellitus – A pilot study

    Science.gov (United States)

    Shanmugasundar, G.; Bhansali, Anil; Walia, Rama; Dutta, Pinaki; Upreti, Vimal

    2012-01-01

    Background & objectives: Conventionally, biphasic human insulin (30/70, BHI) is used twice daily for the management of patients with diabetes. However, this regimen is suboptimal to control post-lunch and/or pre-dinner hyperglycaemia in some patients. This study was undertaken to compare the efficacy and safety of thrice-daily biphasic human insulin (30/70, BHI) versus basal detemir and bolus aspart (BB) in patients with poorly controlled type 2 diabetes mellitus (T2DM). Methods: In this open labelled randomized pilot study, 50 patients with uncontrolled T2DM on twice-daily BHI and insulin sensitizers were randomized either to BHI thrice-daily or BB regimen. HbA1c, six point plasma glucose profile, increment in insulin dose, weight gain, hypoglycaemic episodes and cost were compared between the two treatment groups at the end of 12 wk. Results: Mean HbAlc (±SD) decreased from 9.0±0.9 per cent at randomization to 7.9±0.8 per cent in BHI (P<0.001) and from 9.4±1.3 to 8.2±1.0 per cent in BB regimen (P<0.001) after 12 wk of treatment. The mean (±SEM) weight gain in patients in the BHI regimen was 1.5±0.33 kg compared to 1.4±0.34 kg in the BB regimen. Insulin dose increment at 12 wk was significantly more in the BB regimen 0.46±0.32 U/kg/day compared to 0.15±0.21 U/kg/day in the BHI regimen (P<0.001). The incidence of major as well as minor hypoglycaemic episodes was not different in both the regimen. The BB regimen was more expensive than the BHI regimen (P<0.001). Interpretation & conclusions: The thrice daily biphasic human insulin regimen is non-inferior to the basal bolus insulin analogue regimen in terms efficacy and safety in patients with poorly controlled T2DM. However, these data require further substantiation in large long term prospective studies. PMID:22382187

  18. Clinical experience of switching from biphasic human insulin to biphasic insulin aspart 30 in Indian patients with type 2 diabetes in the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    A K Das

    2015-01-01

    Full Text Available Aim: The aim of the following study is to evaluate the safety and effectiveness of switching from biphasic human insulin (BHI to biphasic insulin aspart 30 (BIAsp 30 in Indian patients with type 2 diabetes as a sub-analysis of the 24-week, non-interventional A 1 chieve study. Materials and Methods: Indian patients switching from BHI to BIAsp 30 based on the physicians′ decisions were included. The primary outcome was the incidence of serious adverse drug reactions (SADRs, including major hypoglycemic events; secondary outcomes included changes in hypoglycemia in the 4 weeks preceding baseline and week 24 and changes from baseline to week 24 in glycated hemoglobin A 1c (HbA 1c , fasting plasma glucose (FPG, postprandial plasma glucose (PPPG, body weight and quality of life (QoL. Results: Overall, 1976 patients (mean ± standard deviation age, 55.1 ± 10.6 years and diabetes duration, 10.1 ± 5.3 years on a mean pre-study BHI dose of 0.44 ± 0.18 U/kg were included. The mean BIAsp 30 dose was 0.43 ± 0.17 U/kg at baseline and 0.44 ± 0.17 U/kg at week 24. No SADRs were reported. The proportion of patients reporting overall hypoglycemic events reduced significantly from baseline to week 24 (15.0% vs. 2.9%, P < 0.0001. The mean HbA 1c level improved significantly from 9.1 ± 1.4% at baseline to 7.5 ± 1.0% at week 24, along with improvements in FPG, post-breakfast PPPG and QoL (P < 0.001. The mean body weight decreased from 69.3 ± 10.8 kg at baseline to 69.1 ± 10.4 kg at week 24 (P = 0.003. Conclusion: Switching from BHI to BIAsp 30 therapy was well-tolerated and was associated with improved glycemic control.

  19. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

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    Freeman Robert S

    2011-02-01

    Full Text Available Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult. Results We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM, we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates

  20. Expression of N-methyl-D-aspartic acid 2A-B and 2B receptors in anterior thalamic nucleus and subiculum complex of rats

    Institute of Scientific and Technical Information of China (English)

    Yuanshan Fu; Xiaokai Ma; Xiaoling Yue; Bin Wang

    2008-01-01

    BACKGROUND: Glutamate acid ionotropic receptor N-methyl-D-aspartic acid (NMDA) takes part in long-term potentiation, thereby influencing the process of learning and memory.OBJECTIVE: To verify expression of NMDA 2A/B and 2B receptors in the anterior thalamic nucleus and subieulum complex of rats.DESIGN, TIME AND SETTING: A single-sample observation was performed at Department of Anatomy in Dalian Mcdical University (Dalian, Liaoning, China) from April to September in 2007.MATERIALS: Ten adult Wistar rats were used for this study, as well as rabbit anti-NMDA 2A/B and 2Bantibodies.METHODS: The rats were anesthetized and perfused, followed by brain resection and coronal sectioning of the brain tissue. A 1:3 series was selected for immunohistochemistry, using antibodies specific to NMDA 2A/B and 2B receptors. Photos were taken using the Nikon image analysis system.MAIN OUTCOME MEASURES: Expression and distribution of immunohistochemistry staining of NMDA 2A/B and 2B receptor subunits.RESULTS: There were a large number of NMDA 2A/B and 2B receptor-positive neurons distributed throughout the anterior dorsal thalamic nucleus. In the anterior ventral thalamic nucleus, distribution of positive neurons was rare, staining intensity was lighter, and cell bodies were smaller compared with the anterior dorsal thalamic nucleus. In the subiculum complex, staining intensity of NMDA 2A/B and 2B-positive neurons was weakest in the molecular layer and stronger in the pyramidal layer, in particular the region with large cell bodies adjacent to the molecular layer. In the multiform layer, more positive neurons of various sizes were detected.CONCLUSION: NMDA 2A/B and 2B receptor subunits were richly distributed in the anterior thalamic nucleus, with a small difference existing between the anterior dorsal nucleus and anterior ventral nucleus.These neurons were also differentially distributed within the three layers of the subiculum complex.