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Sample records for acid decarboxylase inhibitor

  1. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena. PMID:2742363

  2. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  3. Genetics Home Reference: aromatic l-amino acid decarboxylase deficiency

    MedlinePlus

    ... aromatic l-amino acid decarboxylase deficiency aromatic l-amino acid decarboxylase deficiency Enable Javascript to view the expand/ ... PDF Open All Close All Description Aromatic l-amino acid decarboxylase (AADC) deficiency is an inherited disorder that ...

  4. Zymographic detection of cinnamic acid decarboxylase activity.

    PubMed

    Prim, Núria; Pastor, F I Javier; Diaz, Pilar

    2002-11-01

    The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

  5. Cerebellar Ataxia and Glutamic Acid Decarboxylase Antibodies

    PubMed Central

    Ariño, Helena; Gresa-Arribas, Nuria; Blanco, Yolanda; Martínez-Hernández, Eugenia; Sabater, Lidia; Petit-Pedrol, Mar; Rouco, Idoia; Bataller, Luis; Dalmau, Josep O.; Saiz, Albert; Graus, Francesc

    2016-01-01

    IMPORTANCE Current clinical and immunologic knowledge on cerebellar ataxia (CA) with glutamic acid decarboxylase 65 antibodies (GAD65-Abs) is based on case reports and small series with short-term follow-up data. OBJECTIVE To report the symptoms, additional antibodies, prognostic factors, and long-term outcomes in a cohort of patients with CA and GAD65-Abs. DESIGN, SETTING, AND PARTICIPANTS Retrospective cohort study and laboratory investigations at a center for autoimmune neurologic disorders among 34 patients with CA and GAD65-Abs, including 25 with long-term follow-up data (median, 5.4 years; interquartile range, 3.1-10.3 years). MAIN OUTCOMES AND MEASURES Analysis of clinicoimmunologic features and predictors of response to immunotherapy. Immunochemistry on rat brain, cultured neurons, and human embryonic kidney cells expressing GAD65, GAD67, α1-subunit of the glycine receptor, and a repertoire of known cell surface autoantigens were used to identify additional antibodies. Twenty-eight patients with stiff person syndrome and GAD65-Abs served as controls. RESULTS The median age of patients was 58 years (range, 33-80 years); 28 of 34 patients (82%) were women. Nine patients (26%) reported episodes of brainstem and cerebellar dysfunction or persistent vertigo several months before developing CA. The clinical presentation was subacute during a period of weeks in 13 patients (38%). Nine patients (26%) had coexisting stiff person syndrome symptoms. Systemic organ-specific autoimmunities (type 1 diabetes mellitus and others) were present in 29 patients (85%). Twenty of 25 patients with long-term follow-up data received immunotherapy (intravenous immunoglobulin in 10 and corticosteroids and intravenous immunoglobulin or other immunosuppressors in 10), and 7 of them (35%) improved. Predictors of clinical response included subacute onset of CA (odds ratio [OR], 0.50; 95% CI, 0.25-0.99; P = .047) and prompt immunotherapy (OR, 0.98; 95% CI, 0.96-0.99; P = .01). Similar

  6. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  7. Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening

    PubMed Central

    Choi, Jae-Yeon; Lawres, Lauren; Toh, Justin Y.; Voelker, Dennis R.; Ben Mamoun, Choukri

    2016-01-01

    Summary Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity PMID:26585333

  8. Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics.

    PubMed

    Poduch, Ewa; Bello, Angelica M; Tang, Sishi; Fujihashi, Masahiro; Pai, Emil F; Kotra, Lakshmi P

    2006-08-10

    Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

  9. DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.

    PubMed Central

    Kallio, A; McCann, P P; Bey, P

    1982-01-01

    DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased. PMID:6812566

  10. Retinoic acid modulation of ultraviolet light-induced epidermal ornithine decarboxylase activity

    SciTech Connect

    Lowe, N.J.; Breeding, J.

    1982-02-01

    Irradiation of skin with ultraviolet light of sunburn range (UVB) leads to a large and rapid induction of the polyamine biosynthetic enzyme ornithine decarboxylase in the epidermis. Induction of epidermal ornithine decarboxylase also occurs following application of the tumor promoting agent 12-0-tetradecanoylphorbol-13 acetate and topical retinoic acid is able to block both this ornithine decarboxylase induction and skin tumor promotion. In the studies described below, topical application of retinoic acid to hairless mouse skin leads to a significant inhibition of UVB-induced epidermal ornithine decarboxylase activity. The degree of this inhibition was dependent on the dose, timing, and frequency of the application of retinoic acid. To show significant inhibition of UVB-induced ornithine decarboxylase the retinoic acid had to be applied within 5 hr of UVB irradiation. If retinoic acid treatment was delayed beyond 7 hr following UVB, then no inhibition of UVB-induced ornithine decarboxylase was observed. The quantities of retinoic acid used (1.7 nmol and 3.4 nmol) have been shown effective at inhibiting 12-0-tetradecanoyl phorbol-13 acetate induced ornithine decarboxylase. The results show that these concentrations of topical retinoic acid applied either before or immediately following UVB irradiation reduces the UVB induction of epidermal ornithine decarboxylase. The effect of retinoic acid in these regimens on UVB-induced skin carcinogenesis is currently under study.

  11. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  12. Anti-glutamic acid decarboxylase antibody positive neurological syndromes.

    PubMed

    Tohid, Hassaan

    2016-07-01

    A rare kind of antibody, known as anti-glutamic acid decarboxylase (GAD) autoantibody, is found in some patients. The antibody works against the GAD enzyme, which is essential in the formation of gamma aminobutyric acid (GABA), an inhibitory neurotransmitter found in the brain. Patients found with this antibody present with motor and cognitive problems due to low levels or lack of GABA, because in the absence or low levels of GABA patients exhibit motor and cognitive symptoms. The anti-GAD antibody is found in some neurological syndromes, including stiff-person syndrome, paraneoplastic stiff-person syndrome, Miller Fisher syndrome (MFS), limbic encephalopathy, cerebellar ataxia, eye movement disorders, and epilepsy. Previously, excluding MFS, these conditions were calledhyperexcitability disorders. However, collectively, these syndromes should be known as "anti-GAD positive neurological syndromes." An important limitation of this study is that the literature is lacking on the subject, and why patients with the above mentioned neurological problems present with different symptoms has not been studied in detail. Therefore, it is recommended that more research is conducted on this subject to obtain a better and deeper understanding of these anti-GAD antibody induced neurological syndromes. PMID:27356651

  13. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice. PMID:26643381

  14. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  15. Aromatic L-amino acid decarboxylase (AADC) is crucial for brain development and motor functions.

    PubMed

    Shih, De-Fen; Hsiao, Chung-Der; Min, Ming-Yuan; Lai, Wen-Sung; Yang, Chianne-Wen; Lee, Wang-Tso; Lee, Shyh-Jye

    2013-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children. PMID:23940784

  16. Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase.

    PubMed

    Lin, Fengming; Ferguson, Kyle L; Boyer, David R; Lin, Xiaoxia Nina; Marsh, E Neil G

    2015-04-17

    The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

  17. Aromatic L-amino acid decarboxylase deficiency with hyperdopaminuria. Clinical and laboratory findings in response to different therapies.

    PubMed

    Fiumara, A; Bräutigam, C; Hyland, K; Sharma, R; Lagae, L; Stoltenborg, B; Hoffmann, G F; Jaeken, J; Wevers, R A

    2002-08-01

    Aromatic L-amino acid decarboxylase (AADC - E.C. 4.1.1.28) converts L-dopa to dopamine and 5-hydroxytryptophan to serotonin. Inherited deficiency of this enzyme leads to decreased brain levels of these neurotransmitters. Clinically this results in the development of a progressive neurometabolic disorder characterized by severe hypotonia, dystonic and choreoathetoid movements, oculogyric crises, and hypothermia from infancy. Here we describe the clinical, biochemical and molecular details of two affected brothers, one of whom, despite the lack of AADC, presented with hyperdopaminuria. In addition, we detail his reactions to treatment with dopaminergic agonists, monoamine oxidase inhibitors and pyridoxine.

  18. Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

    PubMed

    Kim, Dong Joon; Roh, Eunmiri; Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Yong-Yeon; Pugliese, Angelo; Shim, Jung-Hyun; Chen, Hanyong; Cho, Eun Jin; Kim, Jong-Eun; Kang, Sun Chul; Paul, Souren; Kang, Hee Eun; Jung, Ji Won; Lee, Sung-Young; Kim, Sung-Hyun; Reddy, Kanamata; Yeom, Young Il; Bode, Ann M; Dong, Zigang

    2016-03-01

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. PMID:26676750

  19. Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Hsieh, Ju-Yi; Yang, Jung-Yen; Lin, Chih-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC(50): 0.20 µM) similar to that of AZ-95-228 (IC(50): 0.16 µM), even though a large segment spanning residues 177-228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC(50) values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC(50) values comparable to that of AZ_WT and formed AZ-ODC complexes with K(d,AZ-ODC) values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function. PMID:21931692

  20. Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.

    PubMed

    Viala, Julie P M; Méresse, Stéphane; Pocachard, Bérengère; Guilhon, Aude-Agnès; Aussel, Laurent; Barras, Frédéric

    2011-01-01

    During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.

  1. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease. PMID:26830512

  2. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease.

  3. Treatment of idiopathic parkinsonism with L-dopa in the absence and presence of decarboxylase inhibitors: effects on plasma levels of L-dopa, dopa decarboxylase, catecholamines and 3-O-methyl-dopa.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-05-01

    The effect of levodopa (L-dopa), alone or in combination with a peripheral decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD), dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been studied. In healthy subjects and in patients with parkinsonism plasma ALAAD level fell after administration of L-dopa + benserazide, but returned to previous levels within 90 min. In a cross-sectional study blood was obtained, 2 h after dosing, from 104 patients with idiopathic parkinsonism, divided into four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa + benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4). Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups 3 and 4, indicating that there was induction of ALAAD by the co-administration of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times higher, while plasma DA levels were not different. The DA/L-dopa ratio was decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group 1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence of on-off fluctuations. In a longitudinal study of three patients started on Madopar treatment the induction of plasma ALAAD was found to occur gradually over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and cerebrospinal fluid are required in order to elucidate whether the ALAAD induction by PDI may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena. PMID:2760634

  4. Treatment of idiopathic parkinsonism with L-dopa in the absence and presence of decarboxylase inhibitors: effects on plasma levels of L-dopa, dopa decarboxylase, catecholamines and 3-O-methyl-dopa.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-05-01

    The effect of levodopa (L-dopa), alone or in combination with a peripheral decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD), dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been studied. In healthy subjects and in patients with parkinsonism plasma ALAAD level fell after administration of L-dopa + benserazide, but returned to previous levels within 90 min. In a cross-sectional study blood was obtained, 2 h after dosing, from 104 patients with idiopathic parkinsonism, divided into four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa + benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4). Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups 3 and 4, indicating that there was induction of ALAAD by the co-administration of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times higher, while plasma DA levels were not different. The DA/L-dopa ratio was decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group 1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence of on-off fluctuations. In a longitudinal study of three patients started on Madopar treatment the induction of plasma ALAAD was found to occur gradually over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and cerebrospinal fluid are required in order to elucidate whether the ALAAD induction by PDI may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  5. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    PubMed Central

    Romagnoli, Gabriele; Luttik, Marijke A. H.; Kötter, Peter; Pronk, Jack T.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

  6. Pyruvate decarboxylase from Pisum sativum. Properties, nucleotide and amino acid sequences.

    PubMed

    Mücke, U; Wohlfarth, T; Fiedler, U; Bäumlein, H; Rücknagel, K P; König, S

    1996-04-15

    To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.

  7. Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.

    PubMed Central

    Kierszenbaum, F; Wirth, J J; McCann, P P; Sjoerdsma, A

    1987-01-01

    The capacity of blood (trypomastigote) forms of Trypanosoma cruzi to infect mouse peritoneal macrophages or rat heart myoblasts in vitro was inhibited by treatment of the trypomastigotes with DL-alpha-difluoromethylarginine (F2Me Arg), monofluoromethylagmatine, or (E)-alpha-monofluoromethyl-3-4-dehydroarginine--all irreversible inhibitors of arginine decarboxylase. Similar results were obtained when F2MeArg-treated parasites were incubated with rat heart myoblasts. The inhibitory effects were characterized by marked reductions in both the proportion of infected cells and the number of parasites per 100 host cells. The concentrations of the arginine decarboxylase inhibitors that affected infectivity had no detectable effect on either the concentration or motility of the parasite and, therefore, could not have affected the collision frequency. F2MeArg appeared to inhibit the ability of T. cruzi to penetrate the host cells since the drug had no significant effect on the extent of parasite binding to the surface of the host cells. The inhibitory effect of F2MeArg was markedly reduced or abrogated in the presence of either agmatine or putrescine, as would have been expected if F2MeArg acted by inhibiting arginine decarboxylase. Addition of F2MeArg to macrophage or myoblast cultures immediately after infection or at a time when virtually all of the intracellular parasites had transformed into the multiplicative amastigote form, resulted in a markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host cells and intracellular multiplication. Furthermore, concentrations of the inhibitors tested that affected the parasite did not alter the viability of the host cells, the cellular density of the cultures, or the ability of uninfected myoblasts to grow. Thus, arginine decarboxylase inhibitors may

  8. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Kaur-Sawhney, R.; Galston, A. W.

    1986-01-01

    We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

  9. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

    PubMed

    Tiburcio, A F; Kaur-Sawhney, R; Galston, A W

    1986-01-01

    We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

  10. Chemically-induced formation of an inhibitor of hepatic uroporphyrinogen decarboxylase in inbred mice with iron overload.

    PubMed Central

    Smith, A G; Francis, J E

    1987-01-01

    An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2',3',4'-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase. PMID:3675556

  11. Volatile Organic Compounds Derived from 2-Keto-Acid Decarboxylase in Microcystis aeruginosa

    PubMed Central

    Hasegawa, Masateru; Nishizawa, Akito; Tsuji, Kiyomi; Kimura, Shigenobu; Harada, Ken-ichi

    2012-01-01

    Volatile organic compounds (VOCs), 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol, were detected together with β-cyclocitral from the cyanobacterium Microcystis aeruginosa NIES-843. These alcohols were optimally produced after 35 d of culture, during which nitrate nitrogen in the cultured broth became exhausted. Additionally, these alcohols were definitely produced using the 2-keto-acid decarboxylase (MaKDC) in Microcystis strains. These results suggested that these VOCs from Microcystis are significant for their lifecycle, because these compounds are not produced by any other genus of cyanobacteria. This is the first report of 2-keto-acid decarboxylase producing 3-methyl-1-butanol and 2-phenylethanol by an oxygenic photosynthetic microorganism. PMID:23047148

  12. Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae.

    PubMed

    Richard, Peter; Viljanen, Kaarina; Penttilä, Merja

    2015-01-01

    The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

  13. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. PMID:22718265

  14. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

  15. Tissue and regional distribution of cysteic acid decarboxylase. A new assay method.

    PubMed

    Wu, J Y; Moss, L G; Chen, M S

    1979-04-01

    A sensitive and rapid assay method method for cysteic acid decarboxylase was develped which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of 35S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.

  16. The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Ramachandran, Shaliny; Houry, Walid A

    2011-11-01

    The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH. PMID:21957966

  17. The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Ramachandran, Shaliny; Houry, Walid A

    2011-11-01

    The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.

  18. Snapshot of a Reaction Intermediate: Analysis of Benzoylformate Decarboxylase in Complex with a Benzoylphosphonate Inhibitor

    SciTech Connect

    Brandt, Gabriel S.; Kneen, Malea M.; Chakraborty, Sumit; Baykal, Ahmet T.; Nemeria, Natalia; Yep, Alejandra; Ruby, David I.; Petsko, Gregory A.; Kenyon, George L.; McLeish, Michael J.; Jordan, Frank; Ringe, Dagmar

    2009-04-22

    Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2{alpha}-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2{alpha}-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 {angstrom} (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.

  19. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    SciTech Connect

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario; Mancheño, José M.

    2007-04-01

    The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

  20. Alternating skew deviation in association with anti-glutamic acid decarboxylase antibodies

    PubMed Central

    Farooq, Asim V.; Soin, Ketki; Moss, Heather E.

    2015-01-01

    The presence of an elevated anti-glutamic acid decarboxylase (GAD) antibody level has been associated with a number of eye movement abnormalities, as well as other findings including cerebellar ataxia and insulin dependent diabetes mellitus. Skew deviation in association with anti-GAD antibodies has not been previously reported. Here we report a case of alternating skew deviation along with cerebellar-brainstem signs in a patient with an elevated anti-GAD antibody titer. Follow-up neurologic evaluation after treatment with intravenous immunoglobulin revealed improvement in cerebellar-brainstem signs, while ophthalmic evaluation was stable. PMID:26594078

  1. Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

    DOE PAGES

    Johnson, Christopher W.; Salvachua, Davinia; Khanna, Payal; Smith, Holly; Peterson, Darren J.; Beckham, Gregg T.

    2016-04-22

    The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCAmore » decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.« less

  2. A glutamic acid decarboxylase (CgGAD) highly expressed in hemocytes of Pacific oyster Crassostrea gigas.

    PubMed

    Li, Meijia; Wang, Lingling; Qiu, Limei; Wang, Weilin; Xin, Lusheng; Xu, Jiachao; Wang, Hao; Song, Linsheng

    2016-10-01

    Glutamic acid decarboxylase (GAD), a rate-limiting enzyme to catalyze the reaction converting the excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA), not only functions in nervous system, but also plays important roles in immunomodulation in vertebrates. However, GAD has rarely been reported in invertebrates, and never in molluscs. In the present study, one GAD homologue (designed as CgGAD) was identified from Pacific oyster Crassostrea gigas. The full length cDNA of CgGAD was 1689 bp encoding a polypeptide of 562 amino acids containing a conserved pyridoxal-dependent decarboxylase domain. CgGAD mRNA and protein could be detected in ganglion and hemocytes of oysters, and their abundance in hemocytes was unexpectedly much higher than those in ganglion. More importantly, CgGAD was mostly located in those granulocytes without phagocytic capacity in oysters, and could dynamically respond to LPS stimulation. Further, after being transfected into HEK293 cells, CgGAD could promote the production of GABA. Collectively, these findings suggested that CgGAD, as a GABA synthase and molecular marker of GABAergic system, was mainly distributed in hemocytes and ganglion and involved in neuroendocrine-immune regulation network in oysters, which also provided a novel insight to the co-evolution between nervous system and immune system. PMID:27208883

  3. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  4. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Liu, Yen-Chin; Liu, Yi-Liang; Su, Jia-Yang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities. PMID:21552531

  5. Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

    PubMed Central

    Bhuiya, Mohammad Wadud; Lee, Soon Goo

    2015-01-01

    The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the β2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications. PMID:25862228

  6. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

    PubMed

    Fenalti, Gustavo; Law, Ruby H P; Buckle, Ashley M; Langendorf, Christopher; Tuck, Kellie; Rosado, Carlos J; Faux, Noel G; Mahmood, Khalid; Hampe, Christiane S; Banga, J Paul; Wilce, Matthew; Schmidberger, Jason; Rossjohn, Jamie; El-Kabbani, Ossama; Pike, Robert N; Smith, A Ian; Mackay, Ian R; Rowley, Merrill J; Whisstock, James C

    2007-04-01

    Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

  7. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    PubMed

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

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

    PubMed

    Bakrania, Anita K; Patel, Snehal S

    2015-08-01

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

  9. Conversion of levulinic acid to 2-butanone by acetoacetate decarboxylase from Clostridium acetobutylicum.

    PubMed

    Min, Kyoungseon; Kim, Seil; Yum, Taewoo; Kim, Yunje; Sang, Byoung-In; Um, Youngsoon

    2013-06-01

    In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4-90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a β-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation. PMID:23624707

  10. Immunotherapy-responsive limbic encephalitis with antibodies to glutamic acid decarboxylase.

    PubMed

    Markakis, Ioannis; Alexopoulos, Harry; Poulopoulou, Cornelia; Akrivou, Sofia; Papathanasiou, Athanasios; Katsiva, Vassiliki; Lyrakos, Georgios; Gekas, Georgios; Dalakas, Marinos C

    2014-08-15

    Glutamic acid decarboxylase (GAD) has been recently identified as a target of humoral autoimmunity in a small subgroup of patients with non-paraneoplastic limbic encephalitis (NPLE). We present a patient with NPLE and positive anti-GAD antibodies who showed significant improvement after long-term immunotherapy. A 48-year old female was admitted with a two-year history of anterograde amnesia and seizures. Brain MRI revealed bilateral lesions of medial temporal lobes. Screening for anti-neuronal antibodies showed high anti-GAD titers in both serum and cerebrospinal fluid (CSF) with strong evidence of intrathecal production. The patient received treatment with prednisolone and long-term plasma exchange. During a 12-month follow-up, she exhibited complete seizure remission and an improvement in memory and visuo-spatial skills. Anti-GAD antibodies may serve as a useful marker to identify a subset of NPLE patients that respond to immunoregulatory treatment.

  11. Intrathecal-specific glutamic acid decarboxylase antibodies at low titers in autoimmune neurological disorders.

    PubMed

    Sunwoo, Jun-Sang; Chu, Kon; Byun, Jung-Ick; Moon, Jangsup; Lim, Jung-Ah; Kim, Tae-Joon; Lee, Soon-Tae; Jung, Keun-Hwa; Park, Kyung-Il; Jeon, Daejong; Jung, Ki-Young; Kim, Manho; Lee, Sang Kun

    2016-01-15

    Autoantibodies to glutamic acid decarboxylase (Gad-Abs) are implicated in various neurological syndromes. The present study aims to identify intrathecal-specific GAD-Abs and to determine clinical manifestations and treatment outcomes. Nineteen patients had GAD-Abs in cerebrospinal fluid but not in paired serum samples. Neurological syndromes included limbic encephalitis, temporal lobe epilepsy, cerebellar ataxia, autonomic dysfunction, and stiff-person syndrome. Immunotherapy had beneficial effects in 57.1% of patients, and the patients with limbic encephalitis responded especially well to immunotherapy. Intrathecal-specific antibodies to GAD at low titers may appear as nonspecific markers of immune activation within the central nervous system rather than pathogenic antibodies causing neuronal dysfunction. PMID:26711563

  12. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10

    SciTech Connect

    Karlsen, A.E.; Hagopian, W.A.; Grubin, C.E.; Dube, S.; Disteche, C.M.; Adler, D.A.; Baermeier, H.; Lernmark, A. ); Mathewes, S.; Grant, F.J.; Foster, D. )

    1991-10-01

    Glutamic acid decarboxylase which catalyzes formation of {gamma}-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, the authors describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different form that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows < 65% identify to previously published, highly conserved GAD-1 brain sequences, which show > 96% deduced amino acid sequence homology among the three species.

  13. Cysteine Sulfinic Acid Decarboxylase Regulation: A Role for FXR and SHP in Murine Hepatic Taurine Metabolism

    PubMed Central

    Kerr, Thomas A.; Matsumoto, Yuri; Matsumoto, Hitoshi; Xie, Yan; Hirschberger, Lawrence L.; Stipanuk, Martha H.; Anakk, Sayeepriyadarshini; Moore, David D.; Watanabe, Mitsuhiro; Kennedy, Susan

    2014-01-01

    Background Bile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor15/19 (FGF15/19). Because bile acid synthesis involves amino acid conjugation, we hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids. Aims To investigate CSAD regulation by bile acids and CSAD regulatory mechanisms. Methods Mice were fed a control diet or a diet supplemented with either 0.5% cholate or 2% cholestyramine. To gain mechanistic insight into CSAD regulation, we utilized GW4064 (FXR agonist), FGF19, or T-0901317 (LXR agonist) and Shp−/− mice. Tissue mRNA expression was determined by qRT-PCR. Amino acids were measured by HPLC. Results Mice supplemented with dietary cholate exhibited reduced hepatic CSAD mRNA expression while those receiving cholestyramine exhibited increased hepatic CSAD mRNA expression. Activation of FXR suppressed CSAD mRNA expression whereas hepatic CSAD mRNA expression was increased in Shp−/− mice. Hepatic hypotaurine concentration (the product of CSAD) was higher in Shp−/− mice with a corresponding increase in serum (but not hepatic) taurine-conjugated bile acids. FGF19 administration suppressed hepatic CYP7A1 mRNA but did not change CSAD mRNA expression. LXR activation induced CYP7A1 mRNA yet failed to induce CSAD mRNA expression. Conclusion CSAD mRNA expression is physiologically regulated by bile acids in a feedback fashion via mechanisms involving SHP and FXR but not FGF15/19 or LXR. These novel findings implicate bile acids as regulators of CSAD mRNA via mechanisms shared in part with CYP7A1. PMID:24033844

  14. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Alexopoulos, Eftichia; Zhao, Boyu; El Bakkouri, Majida; Thibault, Guillaume; Liu, Kaiyin; Ramachandran, Shaliny; Snider, Jamie; Pai, Emil F; Houry, Walid A

    2011-03-01

    The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses. PMID:21278708

  15. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-li...

  16. Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

    PubMed

    Bacchi, Cyrus J; Barker, Robert H; Rodriguez, Aixa; Hirth, Bradford; Rattendi, Donna; Yarlett, Nigel; Hendrick, Clifford L; Sybertz, Edmund

    2009-08-01

    Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

  17. Structural perspective on the direct inhibition mechanism of EGCG on mammalian histidine decarboxylase and DOPA decarboxylase.

    PubMed

    Ruiz-Pérez, M Victoria; Pino-Ángeles, Almudena; Medina, Miguel A; Sánchez-Jiménez, Francisca; Moya-García, Aurelio A

    2012-01-23

    Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors. PMID:22107329

  18. Amino acids regulate expression of antizyme-1 to modulate ornithine decarboxylase activity.

    PubMed

    Ray, Ramesh M; Viar, Mary Jane; Johnson, Leonard R

    2012-02-01

    In a glucose-salt solution (Earle's balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5'-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser(2448) and AKT-Ser(473), suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism. PMID:22157018

  19. Selective loss of Purkinje cells in a patient with anti‐glutamic acid decarboxylase antibody‐associated cerebellar ataxia

    PubMed Central

    Ishida, Kazuyuki; Mitoma, Hiroshi; Wada, Yoshiaki; Oka, Teruaki; Shibahara, Junji; Saito, Yuko; Murayama, Shigeo; Mizusawa, Hidehiro

    2007-01-01

    Anti‐glutamic acid decarboxylase antibody is associated with the development of progressive cerebellar ataxia and slowly progressive insulin‐dependent diabetes mellitus. Previously, the neurophysiological characteristics of IgG in the cerebrospinal fluid of a patient with anti‐glutamic acid decarboxylase antibody‐associated progressive cerebellar ataxia and slowly progressive insulin‐dependent diabetes mellitus were reported. Using a voltage‐gated whole‐cell recording technique, it was observed that the IgG in the cerebrospinal fluid of the patient selectively suppressed the inhibitory postsynaptic currents in the Purkinje cells. The patient died from aspiration pneumonia. Postmortem examination showed almost complete depletion of the Purkinje cells with Bergmann gliosis. Therefore, the main cause of cerebellar ataxia observed in this case may be attributed to the near‐complete depletion of the Purkinje cells. In this paper, the pathomechanisms underlying Purkinje cell damage are discussed. PMID:17119008

  20. QSAR study of malonyl-CoA decarboxylase inhibitors using GA-MLR and a new strategy of consensus modeling.

    PubMed

    Li, Jiazhong; Lei, Beilei; Liu, Huanxiang; Li, Shuyan; Yao, Xiaojun; Liu, Mancang; Gramatica, Paola

    2008-12-01

    Quantitative structure-activity relationship (QSAR) of a series of structural diverse malonyl-CoA decarboxylase (MCD) inhibitors have been investigated by using the predictive single model as well as the consensus analysis based on a new strategy proposed by us. Self-organizing map (SOM) neural network was employed to divide the whole data set into representative training set and test set. Then a multiple linear regressions (MLR) model population was built based on the theoretical molecular descriptors selected by Genetic Algorithm using the training set. In order to analyze the diversity of these models, multidimensional scaling (MDS) was employed to explore the model space based on the Hamming distance matrix calculated from each two models. In this space, Q(2) (cross-validated R(2)) guided model selection (QGMS) strategy was performed to select submodels. Then consensus modeling was built by two strategies, average consensus model (ACM) and weighted consensus model (WCM), where each submodel had a different weight according to the contribution of model expressed by MLR regression coefficients. The obtained results prove that QGMS is a reliable and practical method to guide the submodel selection in consensus modeling building and our weighted consensus model (WCM) strategy is superior to the simple ACM.

  1. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

    PubMed Central

    Izzo, Emanuela; Auta, James; Impagnatiello, Francesco; Pesold, Christine; Guidotti, Alessandro; Costa, Erminio

    2001-01-01

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72–96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABAA (γ-aminobutyric acid type A) receptor subunits (decrease in γ2 and α1; increase in α5) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD67. In contrast, dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal. PMID:11248104

  2. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

    PubMed

    Izzo, E; Auta, J; Impagnatiello, F; Pesold, C; Guidotti, A; Costa, E

    2001-03-13

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

  3. Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy

    PubMed Central

    Jasuja, Ravi; Costello, James C; Singh, Rajan; Gupta, Vandana; Spina, Catherine S; Toraldo, Gianluca; Jang, Hyeran; Li, Hu; Serra, Carlo; Guo, Wen; Chauhan, Pratibha; Narula, Navjot S; Guarneri, Tyler; Ergun, Ayla; Travison, Thomas G; Collins, James J; Bhasin, Shalender

    2014-01-01

    Because of its anabolic effects on muscle, testosterone is being explored as a function-promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterone’s adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterone’s promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone’s effects on prostate, but did not affect testosterone’s anabolic effects on muscle. Co-administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate-sparing anabolic therapy. PMID:24305501

  4. Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy.

    PubMed

    Jasuja, Ravi; Costello, James C; Singh, Rajan; Gupta, Vandana; Spina, Catherine S; Toraldo, Gianluca; Jang, Hyeran; Li, Hu; Serra, Carlo; Guo, Wen; Chauhan, Pratibha; Narula, Navjot S; Guarneri, Tyler; Ergun, Ayla; Travison, Thomas G; Collins, James J; Bhasin, Shalender

    2014-04-01

    Because of its anabolic effects on muscle, testosterone is being explored as a function-promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterone's adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterone's promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone's effects on prostate, but did not affect testosterone's anabolic effects on muscle. Co-administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate-sparing anabolic therapy.

  5. Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

    PubMed

    Atwal, Paldeep S; Donti, Taraka R; Cardon, Aaron L; Bacino, C A; Sun, Qin; Emrick, L; Reid Sutton, V; Elsea, Sarah H

    2015-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

  6. Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis.

    PubMed

    Chang, Yen-Chia; Ding, Shih-Torng; Lee, Yen-Hua; Wang, Ya-Ching; Huang, Ming-Feng; Liu, I-Hsuan

    2013-02-01

    Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos. PMID:22907836

  7. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    PubMed

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs.

  8. Spinal cord injury enables aromatic L-amino acid decarboxylase cells to synthesize monoamines.

    PubMed

    Wienecke, Jacob; Ren, Li-Qun; Hultborn, Hans; Chen, Meng; Møller, Morten; Zhang, Yifan; Zhang, Mengliang

    2014-09-01

    Serotonin (5-HT), an important modulator of both sensory and motor functions in the mammalian spinal cord, originates mainly in the raphe nuclei of the brainstem. However, following complete transection of the spinal cord, small amounts of 5-HT remain detectable below the lesion. It has been suggested, but not proven, that this residual 5-HT is produced by intraspinal 5-HT neurons. Here, we show by immunohistochemical techniques that cells containing the enzyme aromatic l-amino acid decarboxylase (AADC) occur not only near the central canal, as reported by others, but also in the intermediate zone and dorsal horn of the spinal gray matter. We show that, following complete transection of the rat spinal cord at S2 level, AADC cells distal to the lesion acquire the ability to produce 5-HT from its immediate precursor, 5-hydroxytryptophan. Our results indicate that this phenotypic change in spinal AADC cells is initiated by the loss of descending 5-HT projections due to spinal cord injury (SCI). By in vivo and in vitro electrophysiology, we show that 5-HT produced by AADC cells increases the excitability of spinal motoneurons. The phenotypic change in AADC cells appears to result from a loss of inhibition by descending 5-HT neurons and to be mediated by 5-HT1B receptors expressed by AADC cells. These findings indicate that AADC cells are a potential source of 5-HT at spinal levels below an SCI. The production of 5-HT by AADC cells, together with an upregulation of 5-HT2 receptors, offers a partial explanation of hyperreflexia below a chronic SCI. PMID:25186745

  9. Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells.

    PubMed

    Miyazaki, Y; Matsufuji, S; Murakami, Y; Hayashi, S

    1993-06-15

    The half-life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC-degrading activity. SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse-transcription/polymerase-chain-reaction (RT-PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441-->Trp replacement near the C-terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC-deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild-type ODC activity decreased with a half-life of 1 h. In-vitro-synthesized mutant ODC with the Cys441-->Trp (or Ala) replacement was also stable in a reticulocyte-lysate ODC-degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441-->Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.

  10. Structural Basis of Enzymatic Activity for the Ferulic Acid Decarboxylase (FADase) from Enterobacter sp. Px6-4

    PubMed Central

    Liang, Lianming; Sun, Yuna; Huang, Jingwen; Li, Xuemei; Cao, Yi; Meng, Zhaohui; Zhang, Ke-Qin

    2011-01-01

    Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an “open-closed” mechanism involving a pocket of 8×8×15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer. PMID:21283705

  11. Wound-Inducible Biosynthesis of Phytoalexin Hydroxycinnamic Acid Amides of Tyramine in Tryptophan and Tyrosine Decarboxylase Transgenic Tobacco Lines1

    PubMed Central

    Guillet, Gabriel; De Luca, Vincenzo

    2005-01-01

    The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC ×TYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine. PMID:15665252

  12. Amino acid decarboxylase activity and other chemical characteristics as related to freshness loss in iced cod (Gadus morhua).

    PubMed

    Hernández-Herrero, M Manuela; Duflos, Guillaume; Malle, Pierre; Bouquelet, Stéphane

    2002-07-01

    Biogenic amine levels and other biochemical indicators were measured to study the safety of and the loss of freshness in iced Atlantic cod. Biogenic amine content exhibited high variability during iced storage of Atlantic cod. Ornithine and lysine decarboxylase activity apparently increased at the end of the storage period. Amino acid activity was probably generated by endogenous amino acid decarboxylases of raw fish. No statistical differences were observed in the total volatile base fraction or in the ammonia or monomethylamine contents during iced storage. However, trimethylamine contents showed a significant exponential relationship with time and sensory score. Cod formed inosine as the major metabolite of IMP. The H and G indices showed a linear relationship with time and sensory score and served as good indicators of cod freshness quality. However, the K, Ki, and P indices showed a logarithmic relationship with time and sensory score. IMP, K, Ki, and P served as indicators of freshness lost during the early stages of chilled storage of cod. PMID:12117250

  13. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89.

    PubMed

    Yuwen, Lei; Zhang, Feng-Li; Chen, Qi-Hua; Lin, Shuang-Jun; Zhao, Yi-Lei; Li, Zhi-Yong

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid sequence of AADC have suggested that L-tryptophan and L-phenylalanine are the potential substrates of AADC. The enzymatic kinetic experiment of the recombinant AADC proved that L-tryptophan is a more reactive substrate of AADC than L-phenylalanine. Meanwhile, the AADC-catalyzed conversion of L-tryptophan into tryptamine was confirmed by means of HPLC and LC/MS. Thus during bacillamide C biosynthesis, the decarboxylation of L-tryptophan to tryptamine is likely conducted first under AADC catalysis, followed by the amidation of tryptamine with the carboxylic product of NRPS gene cluster.

  14. Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

    PubMed

    Takagi, K; Suganuma, M; Kagechika, H; Shudo, K; Ninomiya, M; Muto, Y; Fujiki, H

    1988-01-01

    Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

  15. An organic solvent-tolerant phenolic acid decarboxylase from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic acids to vinyl phenol derivatives.

    PubMed

    Hu, Hongfei; Li, Lulu; Ding, Shaojun

    2015-06-01

    A new phenolic acid decarboxylase gene (blpad) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The full-length blpad encodes a 166-amino acid polypeptide with a predicted molecular mass and pI of 19,521 Da and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37 °C and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic, and sinapic acids at the relative ratios of specific activities 100:74.59:34.41:0.29. Kinetic constant K m values toward p-coumaric, ferulic, caffeic, and sinapic acids were 1.64, 1.55, 1.93, and 2.45 mM, and V max values were 268.43, 216.80, 119.07, and 0.78 U mg(-1), respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvent tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphasic organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinylphenol and 4-vinylguaiacol. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 60.63 g l(-1) 4-vinylphenol and 58.30 g l(-1) 4-vinylguaiacol with the conversion yields 97.02 and 70.96 %, respectively. The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in the bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system. PMID:25547838

  16. Co-localization of glutamic acid decarboxylase and vesicular GABA transporter in cytochrome oxidase patches of macaque striate cortex.

    PubMed

    Adams, Daniel L; Economides, John R; Horton, Jonathan C

    2015-01-01

    The patches in primary visual cortex constitute hot spots of metabolic activity, manifested by enhanced levels of cytochrome oxidase (CO) activity. They are also labeled preferentially by immunostaining for glutamic acid decarboxylase (GAD), γ-aminobutyric acid (GABA), and parvalbumin. However, calbindin shows stronger immunoreactivity outside patches. In light of this discrepancy, the distribution of the vesicular GABA transporter (VGAT) was examined in striate cortex of two normal macaques. VGAT immunoreactivity was strongest in layers 4B, 4Cα, and 5. In tangential sections, the distribution of CO, GAD, and VGAT was compared in layer 2/3. There was a close match between all three labels. This finding indicates that GABA synthesis is enriched in patches, and that inhibitory synapses are more active in patches than interpatches. PMID:26579566

  17. The influence of the cell free solution of lactic acid bacteria on tyramine production by food borne-pathogens in tyrosine decarboxylase broth.

    PubMed

    Toy, Nurten; Özogul, Fatih; Özogul, Yesim

    2015-04-15

    The function of cell-free solutions (CFSs) of lactic acid bacteria (LAB) on tyramine and other biogenic amine production by different food borne-pathogens (FBPs) was investigated in tyrosine decarboxylase broth (TDB) using HPLC. Cell free solutions were prepared from four LAB strains. Two different concentrations which were 50% (5 ml CFS+5 ml medium/1:1) and 25% (2.5 ml CFS+7.5 ml medium/1:3) CFS and the control without CFS were prepared. Both concentration of CFS of Streptococcus thermophilus and 50% CFS of Pediococcus acidophilus inhibited tyramine production up to 98% by Salmonella paratyphi A. Tyramine production by Escherichia coli was also inhibited by 50% CFS of Lactococcus lactis subsp. lactis and 25% CFS of Leuconostoc lactis. subsp. cremoris. The inhibitor effect of 50% CFS of P. acidophilus was the highest on tyramine production (55%) by Listeria monocytogenes, following Lc. lactis subsp. lactis and Leuconostoc mesenteroides subsp. cremoris (20%) whilst 25% CFS of Leu. mes. subsp. cremoris and Lc. lactis subsp. lactis showed stimulator effects (160%). The stimulation effects of 50% CFS of S. thermophilus and Lc. lactis subsp. lactis were more than 70% by Staphylococcus aureus comparing to the control. CFS of LAB strains showed statistically inhibitor effect since lactic acid inhibited microbial growth, decreased pH quickly and reduced the formation of AMN and BAs. Consequently, in order to avoid the formation of high concentrations of biogenic amines in fermented food by bacteria, it is advisable to use CFS for food and food products.

  18. Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species ▿ †

    PubMed Central

    Rude, Mathew A.; Baron, Tarah S.; Brubaker, Shane; Alibhai, Murtaza; Del Cardayre, Stephen B.; Schirmer, Andreas

    2011-01-01

    Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleTJE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family. PMID:21216900

  19. On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein.

    PubMed

    van der Meer, R A; Groen, B W; Duine, J A

    1989-03-27

    Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.

  20. Aromatic amino Acid decarboxylase deficiency not responding to pyridoxine and bromocriptine therapy: case report and review of response to treatment.

    PubMed

    Alfadhel, Majid; Kattan, Rana

    2014-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency (MIM #608643) is an autosomal recessive inborn error of monoamines. It is caused by a mutation in the DDC gene that leads to a deficiency in the AADC enzyme. The clinical features of this condition include a combination of dopamine, noradrenaline, and serotonin deficiencies, and a patient may present with hypotonia, oculogyric crises, sweating, hypersalivation, autonomic dysfunction, and progressive encephalopathy with severe developmental delay. We report the case of an 8-month-old boy who presented with the abovementioned symptoms and who was diagnosed with AADC deficiency based on clinical, biochemical, and molecular investigations. Treatment with bromocriptine and pyridoxine showed no improvement. These data support the findings observed among previously reported cohorts that showed poor response of this disease to current regimens. Alternative therapies are needed to ameliorate the clinical complications associated with this disorder.

  1. Dynamic changes in gamma-aminobutyric acid and glutamate decarboxylase activity in oats (Avena nuda L.) during steeping and germination.

    PubMed

    Xu, Jian Guo; Hu, Qing Ping; Duan, Jiang Lian; Tian, Cheng Rui

    2010-09-01

    Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and provides beneficial effects for human and other animals health. To accumulate GABA, samples from two different naked oat cultivars, Baiyan II and Bayou I, were steeped and germinated in an incubator. The content of GABA and glutamic acid as well as the activity of the glutamate decarboxylase (GAD) in oats during steeping and germination were investigated with an amino acid automatic analyzer. Compared with raw groats, an increase in GABA content of oat groats during steeping and germination was continuously observed for two oat cultivars. The activity of GAD increased greatly at the end of steeping and the second stage of germination for Baiyan II and Bayou I, respectively. Glutamic acid content of treated oat groats was significantly lower than that in raw groats until the later period of germination. GABA was correlated (p<0.01) significantly and positively with the glutamic acid rather than GAD activity in the current study. The results indicates that steeping and germination process under highly controlled conditions can effectively accumulate the GABA in oat groats for Baiyan II and Bayou I, which would greatly facilitate production of nutraceuticals or food ingredients that enable consumers to gain greater access to the health benefits of oats. However, more assays need to be further performed with more oat cultivars.

  2. Molecular and Functional Analyses of Amino Acid Decarboxylases Involved in Cuticle Tanning in Tribolium castaneum*

    PubMed Central

    Arakane, Yasuyuki; Lomakin, Joseph; Beeman, Richard W.; Muthukrishnan, Subbaratnam; Gehrke, Stevin H.; Kanost, Michael R.; Kramer, Karl J.

    2009-01-01

    Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide β-alanine and dopamine used in insect cuticle tanning. β-Alanine is conjugated with dopamine to yield N-β-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of β-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA. PMID:19366687

  3. Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli.

    PubMed

    Kang, Taek Jin; Ho, Ngoc Anh Thu; Pack, Seung Pil

    2013-08-15

    Escherichia coli glutamate decarboxylase (GAD) converts glutamate into γ-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1M GABA in 3h, which can be translated into a space-time yield of 34.3g/L/h.

  4. New Insights into the Design of Inhibitors of Human S-Adenosylmethionine Decarboxylase: Studies of Adenine C[superscript 8] Substitution in Structural Analogues of S-Adenosylmethionine

    SciTech Connect

    McCloskey, Diane E.; Bale, Shridhar; Secrist, III, John A.; Tiwari, Anita; Moss, III, Thomas H.; Valiyaveettil, Jacob; Brooks, Wesley H.; Guida, Wayne C.; Pegg, Anthony E.; Ealick, Steven E.

    2009-04-02

    S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C{sup 8}-substituted adenine analogues bound in the active site.

  5. New Insights into the Design of Inhibitors of Human S-Adenosylmethionine Decarboxylase: Studies of Adenine C8 Substitution in Structural Analogues of S-Adenosylmethionine†

    PubMed Central

    2009-01-01

    S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C8-substituted adenine analogues bound in the active site. PMID:19209891

  6. Heterocyclics as corrosion inhibitors for acid media

    SciTech Connect

    Ajmal, M.; Khan, M.A.W.; Ahmad, S.; Quraishi, M.A.

    1996-12-01

    The available literature on the use of heterocyclic compounds as corrosion inhibitors in acid media has been reviewed. It has been noted that the workers in this field have either used sulfur or nitrogen containing heterocyclic compounds for studying inhibition action. The authors have synthesized compounds containing sulfur and nitrogen both in the same ring and studied their inhibition action in acid media. These compounds were found to be better inhibitors than those containing either atoms alone.

  7. Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography.

    PubMed

    Lee, Seungwoon; Ahn, Jungoh; Kim, Yeon-Gu; Jung, Joon-Ki; Lee, Hongweon; Lee, Eun Gyo

    2013-01-15

    We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.

  8. Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

    PubMed

    Maifreni, Michela; Frigo, Francesca; Bartolomeoli, Ingrid; Innocente, Nadia; Biasutti, Marialuisa; Marino, Marilena

    2013-02-01

    The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica. PMID:23298547

  9. Knockout of the p-Coumarate Decarboxylase Gene from Lactobacillus plantarum Reveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

    PubMed Central

    Barthelmebs, Lise; Divies, Charles; Cavin, Jean-François

    2000-01-01

    Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol · min−1 · mg−1 in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity. PMID:10919793

  10. Hydrogen peroxide-independent production of α-alkenes by OleTJE P450 fatty acid decarboxylase

    PubMed Central

    2014-01-01

    Background Cytochrome P450 OleTJE from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H2O2 as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleTJE fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents. Results We report the H2O2-independent activity of OleTJE for the first time. In the presence of NADPH and O2, this P450 enzyme efficiently decarboxylates long-chain fatty acids (C12 to C20) in vitro when partnering with either the fused P450 reductase domain RhFRED from Rhodococcus sp. or the separate flavodoxin/flavodoxin reductase from Escherichia coli. In vivo, expression of OleTJE or OleTJE-RhFRED in different E. coli strains overproducing free fatty acids resulted in production of variant levels of multiple α-alkenes, with a highest total hydrocarbon titer of 97.6 mg·l-1. Conclusions The discovery of the H2O2-independent activity of OleTJE not only raises a number of fundamental questions on the monooxygenase-like mechanism of this peroxygenase, but also will direct the future metabolic engineering work toward improvement of O2/redox partner(s)/NADPH for overproduction of α-alkenes by OleTJE. PMID:24565055

  11. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

    PubMed

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

    2015-07-01

    Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

  12. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

    PubMed

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

    2015-07-01

    Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products. PMID:25757029

  13. Brain glutamate decarboxylase and pyrroloquinoline quinone.

    PubMed

    Choi, S Y; Khemlani, L S; Churchich, J E

    1992-01-01

    Porcine brain glutamate decarboxylase was examined for the presence of covalently bound pyrroloquinoline quinone (PQQ). HPLC analysis of pure glutamate decarboxylase subjected to the hexanol extraction procedure gave negative results when monitored at 320 nm, the maximum of absorbance of 4-hydroxy-5-hexoxy-PQQ. Resolved glutamate decarboxylase exhibits a structureless absorption band at wavelengths longer than 300 nm which cannot be attributed to PQQ. The holoenzyme is not a pyridoxal-quinoprotein; its catalytic mechanism involves the participation of only one cofactor, i.e. pyridoxal-5-P. Free PQQ is a strong inhibitor of the decarboxylase (Ki = 13 microM) and the reaction with the protein results in spectral changes resembling those of polylysine treated with PQQ. If the concentration of free PQQ in some regions of the brain reaches the micromolar level, then PQQ might play a role in the regulation of glutamate decarboxylase activity.

  14. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

    2014-01-01

    Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu. PMID:24616376

  15. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

    2014-01-01

    Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.

  16. Japanese cases of acute onset diabetic ketosis without acidosis in the absence of glutamic acid decarboxylase autoantibody.

    PubMed

    Iwasaki, Yorihiro; Hamamoto, Yoshiyuki; Kawasaki, Yukiko; Ikeda, Hiroki; Honjo, Sachiko; Wada, Yoshiharu; Koshiyama, Hiroyuki

    2010-04-01

    We report consecutive Japanese patients presented with acute onset diabetic ketosis who had negative glutamic acid decarboxylase autoantibody (GADAb) to clarify the clinical characteristics of them. A total of consecutive 1,296 in-patients with newly diagnosed diabetes mellitus, who were admitted to our center from April 2003 to October 2008, were analyzed. Among them, 17 patients who presented with acute onset diabetic ketosis without acidosis, and found to be negative for GADAb, were included. They showed male preponderance (n = 15). Ten patients had history of excessive ingestion of sugar-containing soft drink. Patients who successfully withdrew insulin therapy by 6 months (n = 7) showed significantly higher insulin secretion capacity and higher body mass index at the time of diagnosis than those who continued insulin therapy at least for 6 months (n = 10). These findings suggest that some of Japanese patients who presented with acute onset diabetic ketosis and negative for GADAb share several clinical characteristics with atypical type 2 diabetes such as ketosis-prone diabetes and "soft-drink ketosis," but others do not. PMID:20960264

  17. Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme.

    PubMed

    Park, D H; Kim, K T; Choi, M U; Samanta, H; Joh, T H

    1992-12-01

    Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.

  18. Chronic social subordination stress modulates glutamic acid decarboxylase (GAD) 67 mRNA expression in central stress circuits

    PubMed Central

    Makinson, Ryan; Lundgren, Kerstin H.; Seroogy, Kim B.; Herman, James P.

    2015-01-01

    Chronic social subordination is a well-known precipitant of numerous psychiatric and physiological health concerns. In this study, we examine the effects of chronic social stress in the visible burrow system (VBS) on the expression of glutamic acid decarboxylase (GAD) 67 and brain-derived neurotropic factor (BDNF) mRNA in forebrain stress circuitry. Male rats in the VBS system form a dominance hierarchy, whereby subordinate males exhibit neuroendocrine and physiological profiles characteristic of chronic exposure to stress. We found that social subordination decreases GAD67 mRNA in the peri-paraventricular nucleus region of the hypothalamus and the interfascicular nucleus of the bed nucleus of the stria terminalis (BNST), and increases in GAD67 mRNA in the hippocampus, medial prefrontal cortex, and dorsal medial hypothalamus. Expression of BDNF mRNA increased in the dorsal region of the BNST, but remained unchanged in all other regions examined. Results from this study indicate that social subordination is associated with several region-specific alterations in GAD67 mRNA expression in central stress circuits, whereas changes in the expression of BDNF mRNA are limited to the BNST. PMID:26066725

  19. Inhibition of ultraviolet-B skin carcinogenesis by all-trans-retinoic acid regimens that inhibit ornithine decarboxylase induction

    SciTech Connect

    Connor, M.J.; Lowe, N.J.; Breeding, J.H.; Chalet, M.

    1983-01-01

    There is a correlation between the ability to induce the polyamine-biosynthetic enzyme ornithine decarboxylase (ODC) and the tumor-promoting ability of various carcinogens in mouse epidermis. Some agents which inhibit skin carcinogenesis also inhibit ODC induction. In this study, all-trans-retinoic acid (RA) regimens that inhibited the induction of epidermal ODC by ultraviolet-B (UVB) were tested for their ability to inhibit UVB skin carcinogenesis. Hairless mice were irradiated once daily with UVB for 20 days, receiving a total dose of UVB (17.1 kJ/sq m). Topical RA was applied immediately (RA, one dose) or applied 0, 1, 2, 3, and 4 hr (RA, five doses) after each irradiance. The mice were maintained for 52 weeks and then sacrificed. Groups treated with RA tended to have fewer mice with tumors, fewer tumors per mouse, smaller tumor diameters, and slower growing tumors than did appropriate irradiated control groups. RA given five times was more effective than was RA given one time at inhibiting UVB skin carcinogenesis. These results show that RA treatments that inhibit epidermal ODC induction may be effective in reducing the carcinogenicity of UVB.

  20. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  1. Japanese cases of acute onset diabetic ketosis without acidosis in the absence of glutamic acid decarboxylase autoantibody.

    PubMed

    Iwasaki, Yorihiro; Hamamoto, Yoshiyuki; Kawasaki, Yukiko; Ikeda, Hiroki; Honjo, Sachiko; Wada, Yoshiharu; Koshiyama, Hiroyuki

    2010-04-01

    We report consecutive Japanese patients presented with acute onset diabetic ketosis who had negative glutamic acid decarboxylase autoantibody (GADAb) to clarify the clinical characteristics of them. A total of consecutive 1,296 in-patients with newly diagnosed diabetes mellitus, who were admitted to our center from April 2003 to October 2008, were analyzed. Among them, 17 patients who presented with acute onset diabetic ketosis without acidosis, and found to be negative for GADAb, were included. They showed male preponderance (n = 15). Ten patients had history of excessive ingestion of sugar-containing soft drink. Patients who successfully withdrew insulin therapy by 6 months (n = 7) showed significantly higher insulin secretion capacity and higher body mass index at the time of diagnosis than those who continued insulin therapy at least for 6 months (n = 10). These findings suggest that some of Japanese patients who presented with acute onset diabetic ketosis and negative for GADAb share several clinical characteristics with atypical type 2 diabetes such as ketosis-prone diabetes and "soft-drink ketosis," but others do not.

  2. Estradiol decreases taurine level by reducing cysteine sulfinic acid decarboxylase via the estrogen receptor-α in female mice liver.

    PubMed

    Ma, Qiwang; Zhao, Jianjun; Cao, Wei; Liu, Jiali; Cui, Sheng

    2015-02-15

    Cysteine sulfinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO) are two rate-limiting enzymes in taurine de novo synthesis, and their expressions are associated with estrogen concentration. The present study was designed to determine the relationship between 17β-estradiol (E₂) and taurine in female mice liver. We initially observed the mice had lower levels of CSAD, CDO, and taurine during estrus than diestrus. We then, respectively, treated the ovariectomized mice, the cultured hepatocytes, and Hep G2 cells with different doses of E₂, and the CSAD and CDO expressions and taurine levels were analyzed. The results showed that E₂ decreased taurine level in the serum and the cultured cells by inhibiting CSAD and CDO expressions. Furthermore, we identified the molecular receptor types through which E₂ plays its role in regulating taurine synthesis, and our results showed that estrogen receptor-α (ERα) expression was much higher than estrogen receptor-β (ERβ) in the liver and hepatocytes, and the inhibiting effects of E₂ on CSAD, CDO, and taurine level were partially abrogated in the ICI-182,780-pretreated liver and hepatocytes, and in ERα knockout mice. These results indicate that estradiol decreases taurine content by reducing taurine biosynthetic enzyme expression in mice liver.

  3. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    PubMed

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  4. Crenarchaeal Arginine Decarboxylase Evolved from an S-Adenosylmethionine Decarboxylase Enzyme*S⃞

    PubMed Central

    Giles, Teresa N.; Graham, David E.

    2008-01-01

    The crenarchaeon Sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. However, genome sequences from S. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5′-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. Instead they have two paralogs of the S-adenosylmethionine decarboxylase (AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both thermostable enzymes self-cleave at conserved serine residues to form amino-terminal β-domains and carboxyl-terminal α-domains with reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine decarboxylation more efficiently than previously studied pyruvoyl enzymes. α-Difluoromethylarginine significantly reduced the ArgDC activity of purified enzyme, and treating growing S. solfataricus cells with this inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the β-subunit of SSO0536 and the α-subunit of SSO0585 had ArgDC activity, implicating residues responsible for substrate specificity in the amino-terminal domain. This crenarchaeal ArgDC is the first example of alternative substrate specificity in the AdoMetDC family. ArgDC activity has evolved through convergent evolution at least five times, demonstrating the utility of this enzyme and the plasticity of amino acid decarboxylases. PMID:18650422

  5. Improvement of the Rett Syndrome Phenotype in a Mecp2 Mouse Model Upon Treatment with Levodopa and a Dopa-Decarboxylase Inhibitor

    PubMed Central

    Szczesna, Karolina; de la Caridad, Olga; Petazzi, Paolo; Soler, Marta; Roa, Laura; Saez, Mauricio A; Fourcade, Stéphane; Pujol, Aurora; Artuch-Iriberri, Rafael; Molero-Luis, Marta; Vidal, August; Huertas, Dori; Esteller, Manel

    2014-01-01

    Rett Syndrome is a neurodevelopmental autism spectrum disorder caused by mutations in the gene coding for methyl CpG-binding protein (MeCP2). The disease is characterized by abnormal motor, respiratory, cognitive impairment, and autistic-like behaviors. No effective treatment of the disorder is available. Mecp2 knockout mice have a range of physiological and neurological abnormalities that resemble the human syndrome and can be used as a model to interrogate new therapies. Herein, we show that the combined administration of Levodopa and a Dopa-decarboxylase inhibitor in RTT mouse models is well tolerated, diminishes RTT-associated symptoms, and increases life span. The amelioration of RTT symptomatology is particularly significant in those features controlled by the dopaminergic pathway in the nigrostratium, such as mobility, tremor, and breathing. Most important, the improvement of the RTT phenotype upon use of the combined treatment is reflected at the cellular level by the development of neuronal dendritic growth. However, much work is required to extend the duration of the benefit of the described preclinical treatment. PMID:24917201

  6. Glutamic acid decarboxylase 65: a link between GABAergic synaptic plasticity in the lateral amygdala and conditioned fear generalization.

    PubMed

    Lange, Maren D; Jüngling, Kay; Paulukat, Linda; Vieler, Marc; Gaburro, Stefano; Sosulina, Ludmila; Blaesse, Peter; Sreepathi, Hari K; Ferraguti, Francesco; Pape, Hans-Christian

    2014-08-01

    An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABAB receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.

  7. Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog.

    PubMed

    Bosma, P T; Blázquez, M; Collins, M A; Bishop, J D; Drouin, G; Priede, I G; Docherty, K; Trudeau, V L

    1999-03-01

    The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD65 and GAD67. These are the products of separate genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a comprehensive phylogenetic analysis, molecular information of GAD forms in other vertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from brains of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldfish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (Coryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebral ganglion plus neural gland of Ciona intestinalis, a protochordate. Whereas GAD65 and GAD67 homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD forms was found in the protochordate. This indicates that the duplication of the vertebrate GAD gene occurred between 400 and 560 million years ago. For both GAD65 and GAD67, the generated phylogenetic tree followed the general tree topology for the major vertebrate classes. In turtle, an alternative spliced form of GAD65, putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from both GAD65 and GAD67, is expressed in C. (N.) armatus. This third form might have originated from an ancient genome duplication specific to modern ray-finned fishes.

  8. Hydrazides of carboxylic acids as inhibitors of steel acidic corrosion

    SciTech Connect

    Aitov, R.G.; Shein, A.B.; Lesnov, A.E.

    1994-09-01

    Hydrazides of carboxylic acids (HCA) inhibit the corrosion of ferrous materials in acids and netral solutions such as stratum and waste waters of oil deposits. In this work, the authors try to explain the above-mentioned difference and to consider HCA as inhibitors of steel hydrogenation.

  9. A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

    PubMed

    Wu, Fang; Christen, Philipp; Gehring, Heinz

    2011-07-01

    Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 μM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest. PMID:21454364

  10. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  11. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass. PMID:26059194

  12. High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase.

    PubMed

    Schwartz, H L; Chandonia, J M; Kash, S F; Kanaani, J; Tunnell, E; Domingo, A; Cohen, F E; Banga, J P; Madec, A M; Richter, W; Baekkeskov, S

    1999-04-16

    The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells. PMID:10222205

  13. Assessment of the effects of glutamic acid decarboxylase antibodies and trace elements on cognitive performance in older adults

    PubMed Central

    Alghadir, Ahmad H; Gabr, Sami A; Al-Eisa, Einas S

    2015-01-01

    Background Homeostatic imbalance of trace elements such as iron (Fe), copper (Cu), and zinc (Zn) demonstrated adverse effects on brain function among older adults. Objective The present study aimed to investigate the effects of trace elements and the presence of anti-glutamic acid decarboxylase antibodies (GADAs) in human cognitive abilities among healthy older adults. Methods A total of 100 healthy subjects (65 males, 35 females; age range; 64–96 years) were recruited for this study. Based on Loewenstein Occupational Therapy Cognitive Assessment (LOTCA) score, the participants were classified according to cognitive performance into normal (n=45), moderate (n=30), and severe (n=25). Cognitive functioning, leisure-time physical activity (LTPA), serum trace elements – Fe, Cu, Zn, Zn/Cu, and GADAs were assessed using LOTCA battery, pre-validated physical activity (PA) questionnaire, atomic absorption, and immunoassay techniques, respectively. Results Approximately 45% of the study population (n=45) had normal distribution of cognitive function and 55% of the study population (n=55) had abnormal cognitive function; they were classified into moderate (score 62–92) and severe (score 31–62). There was a significant reduction in the level of Zn and Zn/Cu ratio along with an increase in the level of Fe, Cu, and anti-GADAs in subjects of severe (P=0.01) and moderate (P=0.01) cognitive performance. LOTCA-cognitive scores correlated positively with sex, HbA1c, Fe, Cu, Zn, and Zn/Cu ratio, and negatively with age, PA, body mass index, and anti-GADAs. Significant inter-correlation was reported between serum trace element concentrations and anti-GADAs which suggest producing a cognitive decline via oxidative and neural damage mechanism. Conclusion This study found significant associations among trace elements, anti-GADAs, and cognitive function in older adults. The homeostatic balance of trace elements should be recommended among older adults for better cognitive

  14. Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse.

    PubMed

    Liu, W S; Pesold, C; Rodriguez, M A; Carboni, G; Auta, J; Lacor, P; Larson, J; Condie, B G; Guidotti, A; Costa, E

    2001-03-13

    Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

  15. Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

    PubMed Central

    Liu, Wen Sheng; Pesold, Christine; Rodriguez, Miguel A.; Carboni, Giovanni; Auta, James; Lacor, Pascal; Larson, John; Condie, Brian G.; Guidotti, Alessandro; Costa, Erminio

    2001-01-01

    Heterozygous reeler mice (HRM) haploinsufficient for reelin express ≈50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD67)-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD67 down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD67 knockout mice (HG67M). These mice exhibited a down-regulation of GAD67 mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients. PMID:11248103

  16. Penicillin inhibitors of purple acid phosphatase.

    PubMed

    Faridoon; Hussein, Waleed M; Ul Islam, Nazar; Guddat, Luke W; Schenk, Gerhard; McGeary, Ross P

    2012-04-01

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

  17. Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

    PubMed

    Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

    2016-05-10

    The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD. PMID:27082660

  18. Raman micro-spectroscopic investigation of the interaction of cultured HCT116 colon cancer cells with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase

    NASA Astrophysics Data System (ADS)

    Akyuz, S.; Ozel, A. E.; Balci, K.; Akyuz, T.; Coker, A.; Arisan, E. D.; Palavan-Unsal, N.; Ozalpan, A.

    2011-05-01

    The interaction of cultured colon cancer cells with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been investigated, using Raman micro-spectroscopy, in order to investigate DFMO induced effects. Raman spectra of the cultured HCT116 colon cancer cells treated with DFMO at different concentrations (0, 1, 2.5, 5, and 7.5 mM) were recorded in the range 550-2300 cm -1. It has been shown that second derivative profile of the raw Raman spectrum of the colon cancer cells (i.e., the original experimental spectrum without any computational corrections) discriminates the weak but sharp bands from the strong, broad fluorescence background, and gives information about the position of the peaks and their band widths. The relative integrated intensities of the 781 cm -1 and 788 cm -1 DNA/RNA marker bands to that of 1451 cm -1 band are found to decrease by addition of DFMO. Up to 65% reduction in the magnitude of the 1003 cm -1 band, the characteristic phenylalanine ring breathing mode, in comparison to that of 1451 band, is observed. The results indicate DFMO induced apoptosis. On the other hand the intensity ratio of the tyrosine Fermi doubled around 830 cm -1 and 850 cm -1, which is a marker of hydrogen-bonding state of phenolic OH, is changed. The addition of DFMO may alter the tyrosine environment in cells, and parts of tyrosine residues are exposed. We also observed some modifications in amide I band, pointing out the alterations of the secondary structure of cell proteins by the presence of DFMO.

  19. Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-11-12

    The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

  20. HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

    PubMed

    Roy, Ajit; Ranjan, Akash

    2016-02-23

    Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family. PMID:26818787

  1. Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: Implications for schizophrenia

    PubMed Central

    Curley, Allison A.; Eggan, Stephen M.; Lazarus, Matt S.; Huang, Z. Josh; Volk, David W.; Lewis, David A.

    2012-01-01

    Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PVGAD67+/−) or in all interneurons (GABAGAD67+/−) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PVGAD67+/− mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABAGAD67+/− mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell typespecificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. PMID:23103418

  2. Glutamic acid decarboxylase (anti-GAD) & tissue transglutaminase (anti-TTG) antibodies in patients with thyroid autoimmunity

    PubMed Central

    Marwaha, R.K.; Garg, M.K.; Tandon, N.; Kanwar, Ratnesh; Narang, A.; Sastry, A.; Saberwal, A.; Bhadra, Kuntal

    2013-01-01

    Background & objectives: Several autoimmune disorders have been reported to be associated with autoimmune thyroiditis and may coexist with other organ-specific autoantibodies. The aim of the present study was to evaluate the presence of tissue transglutaminase (anti-TTG) and glutamic acid decarboxylase (anti-GAD) antibodies in patients suffering from autoimmune thyroiditis as diagnosed by anti-thyroid peroxidase (anti-TPO) antibodies, which may indicate high risk for developing celiac disease or type 1 diabetes mellitus. Methods: Five thousand children and 2800 adults were screening as part of a general health examination done on a voluntary basis in four different parts of Delhi. A total of 577 subjects positive for anti-TPO antibody constituted the cases. Equal number of age and sex matched anti-TPO antibody negative controls were randomly selected from the same cohort to form paired case control study. The cases and controls were further divided into two groups as follows: group-1 (children and adolescent <18 yr), group-2 (adults >18 yr). Serum samples of cases and controls were analysed for thyroid function test (FT3, FT4, and TSH), anti-TTG and anti-GAD antibodies. Results: A total of 1154 subjects (577 cases and 577 controls) were included in this study. Hypothyroidism was present in 40.2 per cent (232) cases compared to only 4.7 per cent (27) in controls (P<0.001). Anti-TTG and anti-GAD antibodies were present in 6.9 and 12.5 per cent subjects among cases compared to 3.5 per cent (P=0.015) and 4.3 per cent (P=0.001) in controls, respectively. Only anti-GAD antibody were significantly positive in cases among children and adolescents (P =0.0044) and adult (P=0.001) compared to controls. Levels of anti-TTG and anti-GAD antibodies increased with increasing titre of anti-TPO antibody. Interpretation & conclusions: Our findings showed high positivity of anti-GAD and anti-TTG antibodies among subjects with thyroid autoimmunity. It is, therefore, important to have

  3. Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus.

    PubMed

    González-Flores, Oscar; Gómora-Arrati, Porfirio; García-Juárez, Marcos; Miranda-Martínez, Alfredo; Armengual-Villegas, Alejandra; Camacho-Arroyo, Ignacio; Guerra-Araiza, Christian

    2011-10-01

    Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A+PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A+B antisense (PR A+B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A+B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A+B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the

  4. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

    PubMed

    Kuo, D J; Dikdan, G; Jordan, F

    1986-03-01

    A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

  5. Elimination of islet cell antibodies and glutamic acid decarboxylase antibodies II in a patient with newly diagnosed insulin-dependent diabetes mellitus.

    PubMed

    Richter, W O; Donner, M G; Schwandt, P

    1997-01-01

    Islet cell antibodies and glutamic acid decarboxylase II (GAD II) antibodies have been discussed in the autoimmune pathogenesis of insulin-dependent diabetes mellitus (IDDM). Hence, immunosuppressants, intravenous immunoglobulins, and plasmapheresis have been used in an effort to modulate autoimmune activity and thereby prevent the destruction of pancreatic beta-cells. We describe the autoantibody (islet cell antibody and GAD II) kinetics and clinical course in a patient with newly diagnosed IDDM treated with a specific immunoglobulin apheresis technique. Five days after the initial diagnosis a 37-year-old patient with IDDM underwent a series of seven immunoglobulin aphereses. Immunoglobulin (IgG, IgA, IgM), islet cell antibody, GAD II, and C-peptide concentrations were monitored for a time course of 74 days. Daily insulin requirements were recorded. One single immunoglobulin apheresis decreased IgG by 66.2 +/- 9.1%, IgA by 66.8 +/- 8.7%, and IgM by 57.7 +/- 12.9%. GAD II antibodies were reduced by 61.9 +/- 12.4%. The islet cell antibody titer declined from 1:32 to 1:4 after the treatment series. There were no relevant changes in the safety parameters determined nor were there any clinical side effects. The efficient decrease in islet cell antibodies and glutamic acid decarboxylase II antibodies in a patient with IDDM encourages further investigations into the impact of this treatment on the clinical course of this autoimmune disorder.

  6. Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses.

    PubMed

    Mei, Xin; Chen, Yiyong; Zhang, Lingyun; Fu, Xiumin; Wei, Qing; Grierson, Don; Zhou, Ying; Huang, Yahui; Dong, Fang; Yang, Ziyin

    2016-01-01

    γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage. PMID:27021285

  7. Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses

    PubMed Central

    Mei, Xin; Chen, Yiyong; Zhang, Lingyun; Fu, Xiumin; Wei, Qing; Grierson, Don; Zhou, Ying; Huang, Yahui; Dong, Fang; Yang, Ziyin

    2016-01-01

    γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage. PMID:27021285

  8. 4-Vinylphenol biosynthesis from cellulose as the sole carbon source using phenolic acid decarboxylase- and tyrosine ammonia lyase-expressing Streptomyces lividans.

    PubMed

    Noda, Shuhei; Kawai, Yoshifumi; Tanaka, Tsutomu; Kondo, Akihiko

    2015-03-01

    Streptomyces lividans was adopted as a host strain for 4-vinylphenol (4VPh) production directly from cellulose. In order to obtain novel phenolic acid decarboxylase (PAD) expressed in S. lividans, PADs distributed among Streptomyces species were screened. Three novel PADs, derived from Streptomycessviceus, Streptomyceshygroscopicus, and Streptomycescattleya, were successfully obtained and expressed in S. lividans. S. sviceus PAD (SsPAD) could convert p-hydroxycinnamic acid (pHCA) to 4VPh more efficiently than the others both in vitro and in vivo. For 4VPh production directly from cellulose, l-tyrosine ammonia lyase derived from Rhodobacter sphaeroides and SsPAD were introduced into endoglucanase-secreting S. lividans, and the 4VPh biosynthetic pathway was constructed therein. The created transformants successfully produced 4VPh directly from cellulose.

  9. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

    NASA Technical Reports Server (NTRS)

    D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

    1987-01-01

    Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

  10. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

    PubMed Central

    De Luca, V; Marineau, C; Brisson, N

    1989-01-01

    The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases. Images PMID:2704736

  11. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

    Dung Pham, Van; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

  12. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

    PubMed

    Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2015-08-10

    Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

  13. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

    PubMed

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

    2014-07-01

    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

  14. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    PubMed

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  15. Increased levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurons after rapid eye movement sleep deprivation in rats.

    PubMed

    Majumdar, S; Mallick, B N

    2003-03-01

    Norepinephrine, acetylcholine and GABA levels alter during rapid eye movement (REM) sleep and its deprivation. Increased synthesis of those neurotransmitters is necessary for their sustained release. Hence, in this study, the concentrations of tyrosine hydroxylase (TH), choline acetyl transferase (ChAT) and glutamic acid decarboxylase (GAD), the enzymes responsible for their synthesis, were immunohistochemically estimated within the neurons in locus coeruleus, laterodorsal tegmentum and pedunculopontine tegmentum and medial preoptic area in REM sleep deprived and control rats. It was observed that as compared to controls, deprivation increased TH and GAD significantly in the locus coeruleus only, while in other areas, they remained unchanged. The findings help explaining the mechanism of increase in neurotransmitter levels in the brain after REM sleep deprivation and their significance has been discussed.

  16. Novel inhibitors of fatty acid amide hydrolase.

    PubMed

    Sit, S Y; Conway, Charlie; Bertekap, Robert; Xie, Kai; Bourin, Clotilde; Burris, Kevin; Deng, Hongfeng

    2007-06-15

    A class of bisarylimidazole derivatives are identified as potent inhibitors of the enzyme fatty acid amide hydrolase (FAAH). Compound 17 (IC(50)=2 nM) dose-dependently (0.1-10mg/kg, iv) potentiates the effects of exogenous anandamide (1 mg/kg, iv) in a rat thermal escape test (Hargreaves test), and shows robust antinociceptive activity in animal models of persistent (formalin test) and neuropathic (Chung model) pain. Compound 17 (20 mg/kg, iv) demonstrates activity in the formalin test that is comparable to morphine (3mg/kg, iv), and is dose-dependently inhibited by the CB1 antagonist SR141716A. In the Chung model, compound 17 shows antineuropathic effects similar to high-dose (100 mg/kg) gabapentin. FAAH inhibition shows potential utility for the clinical treatment of persistent and neuropathic pain.

  17. Development of Inhibitors of Salicylic Acid Signaling.

    PubMed

    Jiang, Kai; Kurimoto, Tetsuya; Seo, Eun-kyung; Miyazaki, Sho; Nakajima, Masatoshi; Nakamura, Hidemitsu; Asami, Tadao

    2015-08-19

    Salicylic acid (SA) plays important roles in the induction of systemic acquired resistance (SAR) in plants. Determining the mechanism of SAR will extend our understanding of plant defenses against pathogens. We recently reported that PAMD is an inhibitor of SA signaling, which suppresses the expression of the pathogenesis-related PR genes and is expected to facilitate the understanding of SA signaling. However, PAMD strongly inhibits plant growth. To minimize the side effects of PAMD, we synthesized a number of PAMD derivatives, and identified compound 4 that strongly suppresses the expression of the PR genes with fewer adverse effects on plant growth than PAMD. We further showed that the adverse effects on plant growth were partially caused the stabilization of DELLA, which is also related to the pathogen responses. These results indicate that compound 4 would facilitate our understanding of SA signaling and its cross talk with other plant hormones.

  18. NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

    PubMed

    Dugast, C; Weber, M J

    2001-04-18

    We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter. PMID:11311976

  19. Loss of Autonoetic Awareness of Recent Autobiographical Episodes and Accelerated Long-Term Forgetting in a Patient with Previously Unrecognized Glutamic Acid Decarboxylase Antibody Related Limbic Encephalitis

    PubMed Central

    Witt, Juri-Alexander; Vogt, Viola Lara; Widman, Guido; Langen, Karl-Josef; Elger, Christian Erich; Helmstaedter, Christoph

    2015-01-01

    We describe a 35-year-old male patient presenting with depressed mood and emotional instability, who complained about severe anterograde and retrograde memory deficits characterized by accelerated long-term forgetting and loss of autonoetic awareness regarding autobiographical memories of the last 3 years. Months before he had experienced two breakdowns of unknown etiology giving rise to the differential diagnosis of epileptic seizures after various practitioners and clinics had suggested different etiologies such as a psychosomatic condition, burnout, depression, or dissociative amnesia. Neuropsychological assessment indicated selectively impaired figural memory performance. Extended diagnostics confirmed accelerated forgetting of previously learned and retrievable verbal material. Structural imaging showed bilateral swelling and signal alterations of temporomesial structures (left >right). Video-EEG monitoring revealed a left temporal epileptic focus and subclincal seizure, but no overt seizures. Antibody tests in serum and liquor were positive for glutamic acid decarboxylase antibodies. These findings led to the diagnosis of glutamic acid decarboxylase antibody related limbic encephalitis. Monthly steroid pulses over 6 months led to recovery of subjective memory and to intermediate improvement but subsequent worsening of objective memory performance. During the course of treatment, the patient reported de novo paroxysmal non-responsive states. Thus, antiepileptic treatment was started and the patient finally became seizure free. At the last visit, vocational reintegration was successfully in progress. In conclusion, amygdala swelling, retrograde biographic memory impairment, accelerated long-term forgetting, and emotional instability may serve as indicators of limbic encephalitis, even in the absence of overt epileptic seizures. The monitoring of such patients calls for a standardized and concerted multilevel diagnostic approach with repeated assessments

  20. Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation.

    PubMed

    Burtin, D; Martin-Tanguy, J; Paynot, M; Rossin, N

    1989-01-01

    We studied the effects of dl-alpha-difluoromethylarginine (DFMA) and dl-alpha-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.

  1. Effects of the Suicide Inhibitors of Arginine and Ornithine Decarboxylase Activities on Organogenesis, Growth, Free Polyamine and Hydroxycinnamoyl Putrescine Levels in Leaf Explants of Nicotiana Xanthi n.c. Cultivated in Vitro in a Medium Producing Callus Formation

    PubMed Central

    Burtin, Daniel; Martin-Tanguy, Josette; Paynot, Michel; Rossin, Nadia

    1989-01-01

    We studied the effects of dl-α-difluoromethylarginine (DFMA) and dl-α-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines. Images Figure 1 Figure 2 PMID:16666499

  2. Locomotor response to L-DOPA in reserpine-treated rats following central inhibition of aromatic L-amino acid decarboxylase: further evidence for non-dopaminergic actions of L-DOPA and its metabolites.

    PubMed

    Alachkar, Amal; Brotchie, Jonathan M; Jones, Owen T

    2010-09-01

    L-DOPA is the most widely used treatment for Parkinson's disease. The anti-parkinsonian and pro-dyskinetic actions of L-DOPA are widely attributed to its conversion, by the enzyme aromatic L-amino acid decarboxylase (AADC), to dopamine. We investigated the hypothesis that exogenous L-DOPA can induce behavioural effects without being converted to dopamine in the reserpine-treated rat-model of Parkinson's disease. A parkinsonian state was induced with reserpine (3 mg/kg s.c.). Eighteen hours later, the rats were administered L-DOPA plus the peripherally acting AADC inhibitor benserazide (25 mg/kg), with or without the centrally acting AADC inhibitor NSD1015 (100 mg/kg). L-DOPA/benserazide alone reversed reserpine-induced akinesia (4158+/-1125 activity counts/6 h, cf vehicle 1327+/-227). Addition of NSD1015 elicited hyperactive behaviour that was approximately 7-fold higher than L-DOPA/benserazide (35755+/-5226, P<0.001). The hyperactivity induced by L-DOPA and NSD1015 was reduced by the alpha(2C) antagonist rauwolscine (1 mg/kg) and the 5-HT(2C) agonist MK212 (5 mg/kg), but not by the D2 dopamine receptor antagonist remoxipride (3 mg/kg) or the D1 dopamine receptor antagonist SCH23390 (1 mg/kg). These data suggest that L-DOPA, or metabolites produced via routes not involving AADC, might be responsible for the generation of at least some L-DOPA actions in reserpine-treated rats. PMID:20542064

  3. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde

    PubMed Central

    Tieman, Denise; Taylor, Mark; Schauer, Nicolas; Fernie, Alisdair R.; Hanson, Andrew D.; Klee, Harry J.

    2006-01-01

    An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADC1A or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2-phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene. PMID:16698923

  4. Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Ko, Ji-seun; Park, Si Jae; Lee, Seung Hwan; Hong, Soon Ho

    2013-08-01

    Gamma-aminobutyric acid (GABA) is a precursor of one of the most promising heat-resistant biopolymers, Nylon-4, and can be produced by the decarboxylation of monosodium glutamate (MSG). In this study, a synthetic protein complex was applied to improve the GABA conversion in engineered Escherichia coli. Complexes were constructed by assembling a single protein-protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/GABA antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue. When GadA and GadC were co-overexpressed via the C-terminus complex, a GABA concentration of 5.65 g/l was obtained from 10 g/l MSG, which corresponds to a GABA yield of 93 %. A significant increase of the GABA productivity was also observed where the GABA productivity increased 2.5-fold in the early culture period due to the introduction of the synthetic protein complex. The GABA pathway efficiency and GABA productivity were enhanced by the introduction of the complex between Gad and glutamate/GABA antiporter.

  5. Glutamic acid decarboxylase activity is stimulated in quail retina neuronal cells transformed by Rous sarcoma virus and is regulated by pp60v-src.

    PubMed Central

    Crisanti, P; Lorinet, A M; Calothy, G; Pessac, B

    1985-01-01

    Rous sarcoma virus (RSV) stimulates in quail embryo neuro-retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY-68, a thermodependent transformation-defective mutant of RSV, stimulation of GAD activity is regulated by pp60v-src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY-68-transformed established NR cell lines, have a high GAD activity which is regulated by pp60v-src, while other clones have a low GAD activity apparently not regulated by pp60v-src. These data indicate that pp60v-src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v-mil. PMID:2992933

  6. [Critical amino acids of ornitin decarboxylase degron: the presence and C-terminal arrangement is insufficient for alfa-fetoprotein degradation].

    PubMed

    Morozov, A V; Timofeev, A V; Morozov, V A; Karpov, V L

    2011-01-01

    Mouse ornithine decarboxylase (ODC) degrades in proteasome in an ubiquitin-independent manner with an averagehalf-life of 2 h. The 37 amino acid long C-terminal fragment known as a degradation signal (degron) is responsible for the effective degradation of ODC. Recently, amino acids being critical for degradation in the ODC-degron have been mapped. Mutations of Cys441 and Ala442 led to protein stabilization, while a substitution of other amino acids composing ODC-degron had almost no effect on the protein turnover; whereas insertions or deletions in region between Ala442 and ODC C-terminus diminished greatly rate of protein degradation, e.g. positioning of the key amino acids from the C-terminus was shown to be crucial. Using these data we introduced both key amino acids into the alfa-fetoprotein with truncated exportation signal (deltaAFP), at the same distance from the C-terminus as they being in the ODC (deltaAFPCAG and deltaAFPLCAG). Removal of N-terminal exportation signal prevented secretion of modified proteins. Using in silico approach we demonstrated no significant difference in hydrophobicity or secondary structure between C-terminus of deltaAFP and mutated proteins. The degradation kinetics of deltaAFP, deltaAFPCAG, deltaAFPLCAG in cyloheximide-chase and proteasome inhibition assay (using MG132) was identical. Obtained results suggest that introduced substitutions are insufficient for effective recognition of mutated deltaAFP by26S proteasome. We assume thatadditional amino aci ds composing ODC-degron or their combine action could also affect degradation. Besides that, one cannot exclude that conformation of the mutated deltaAFP limits its C-terminus accessibility to proteasome. PMID:21790016

  7. Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis

    SciTech Connect

    Sumi-Ichinose, Chiho ); Ichinose, Hiroshi; Nagatsu, Toshiharu ); Takahashi, Eiichi; Hori, Tadaaki )

    1992-03-03

    Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. The authors proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5{prime} region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position {minus}111 by primer extension and S1 mapping. There were no typical TATA box' and CAAT box' within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

  8. Exponential increase of glutamic acid decarboxylase (GAD) antibody titer after initiating and stopping insulin in a patient with slowly progressive type 1 diabetes.

    PubMed

    Nishimura, Akihiro; Nagasawa, Kaoru; Okubo, Minoru; Kobayashi, Tetsuro; Mori, Yasumichi

    2015-01-01

    Few articles have described fluctuations in glutamic acid decarboxylase antibody (GADAb) levels after a diagnosis of slowly progressive type 1 diabetes (SPIDDM). Here, we present a case in which GADAb levels exponentially increased after initiating and stopping insulin. A 64-year-old female patient newly diagnosed with SPIDDM was admitted and started multiple daily insulin injections. The patient's GADAb titer was 6.9 U/mL (normal: <1.4 U/mL) and the patient had a type 1 diabetes susceptible HLA class II haplotype known in the Japanese population as: DRB1*04:05-DQB1*04:01. When the patient's "honeymoon period" set in, hypoglycemia was observed and the dose of insulin was reduced. Two months after the diagnosis, 1 unit of insulin glargine/day was being injected and the patient demonstrated good glycemic control. Subsequently, the patient's home doctor recommended that insulin injections be stopped. Three months after the diagnosis, the patient's GADAb titer suddenly increased to 1600 U/mL. The patient's GADAb titer decreased but was still positive (40 U/mL) 36 months after diagnosis. HbA1c levels were maintained below 7%, and oral glucose tolerance tests at 10, 26, and 36 months after diagnosis suggested that the patient had preserved insulin secretion. To the best of our knowledge, this is the first report that describes exponential increases in GADAb after initiating and stopping insulin in a patient with SPIDDM.

  9. Characterization of CD4+ T cells specific for glutamic acid decarboxylase (GAD65) and proinsulin in a patient with stiff-person syndrome but without type 1 diabetes

    PubMed Central

    Hänninen, Arno; Soilu-Hänninen, Merja; Hampe, Christiane S.; Deptula, Angie; Geubtner, Kelly; Ilonen, Jorma; Knip, Mikael; Reijonen, Helena

    2010-01-01

    Glutamic acid decarboxylase GAD is a rate-limiting enzyme in the synthesis of GABA and an important autoantigen in both type 1 diabetes (T1D) and in stiff-person syndrome (SPS). Autoantibodies (GADA) to the 65kD isoform of GAD are a characteristic feature in both diseases. Approximately 30% of SPS patients develop diabetes, yet, it is unclear to which extent co-existing autoimmunity to GAD65 and other islet autoantigens determines the risk of developing T1D. In this study we monitored CD4+ T-cell responses to GAD65 and proinsulin in a patient with SPS who remained normoglycemic during the 46-month follow-up. Fluctuating but persistent T-cell reactivity to GAD65 was identified, as well as T-cell reactivity to proinsulin at one time point. The majority of the T-cell clones isolated from the SPS patient produced high levels of Th2 cytokines (IL-13, IL-5 and IL-4). We also examined levels of GADA, insulin and IA-2 autoantibodies, and epitope specificity of GADA. In both serum and cerebrospinal fluid GADA levels were high, and GADA persisted throughout the follow-up. Despite T-cell reactivity to both GAD65 and proinsulin, autoantibodies to other islet autoantigens did not develop. Further follow-up will determine whether or not the beta-cell autoimmunity observed in this patient will eventually lead to T1D. PMID:20503259

  10. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and substance P in neural areas mediating motion-induced emesis: Effects of vagal stimulation on GAD immunoreactivity

    NASA Technical Reports Server (NTRS)

    Damelio, F.; Gibbs, M. A.; Mehler, W. R.; Daunton, Nancy G.; Fox, Robert A.

    1991-01-01

    Immunocytochemical methods were employed to localize the neurotransmitter amino acid gamma-aminobutyric acid (GABA) by means of its biosynthetic enzyme glutamic acid decarboxylase (GAD) and the neuropeptide substance P in the area postrema (AP), area subpostrema (ASP), nucleus of the tractus solitarius (NTS), and gelatinous nucleus (GEL). In addition, electrical stimulation was applied to the night vagus nerve at the cervical level to assess the effects on GAD-immunoreactivity (GAR-IR). GAD-IR terminals and fibers were observed in the AP, ASP, NTS, and GEL. They showed pronounced density at the level of the ASP and gradual decrease towards the solitary complex. Nerve cells were not labelled in our preparations. Ultrastructural studies showed symmetric or asymmetric synaptic contracts between labelled terminals and non-immunoreactive dendrites, axons, or neurons. Some of the labelled terminals contained both clear- and dense-core vesicles. Our preliminary findings, after electrical stimulation of the vagus nerve, revealed a bilateral decrease of GAD-IR that was particularly evident at the level of the ASP. SP-immunoreactive (SP-IR) terminals and fibers showed varying densities in the AP, ASP, NTS, and GEL. In our preparations, the lateral sub-division of the NTS showed the greatest accumulation. The ASP showed medium density of immunoreactive varicosities and terminals and the AP and GEL displayed scattered varicose axon terminals. The electron microscopy revealed that all immunoreactive terminals contained clear-core vesicles which make symmetric or asymmetric synaptic contact with unlabelled dendrites. It is suggested that the GABAergic terminals might correspond to vagal afferent projections and that GAD/GABA and substance P might be co-localized in the same terminal allowing the possibility of a regulated release of the transmitters in relation to demands.

  11. High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kuroiwa, Mari; Kino, Kuniki

    2015-05-25

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L(-1)) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

  12. Mammalian Dopa decarboxylase: structure, catalytic activity and inhibition.

    PubMed

    Bertoldi, Mariarita

    2014-03-15

    Mammalian Dopa decarboxylase catalyzes the conversion of L-Dopa and L-5-hydroxytryptophan to dopamine and serotonin, respectively. Both of them are biologically active neurotransmitters whose levels should be finely tuned. In fact, an altered concentration of dopamine is the cause of neurodegenerative diseases, such as Parkinson's disease. The chemistry of the enzyme is based on the features of its coenzyme pyridoxal 5'-phosphate (PLP). The cofactor is highly reactive and able to perform multiple reactions, besides decarboxylation, such as oxidative deamination, half-transamination and Pictet-Spengler cyclization. The structure resolution shows that the enzyme has a dimeric arrangement and provides a molecular basis to identify the residues involved in each catalytic activity. This information has been combined with kinetic studies under steady-state and pre-steady-state conditions as a function of pH to shed light on residues important for catalysis. A great effort in DDC research is devoted to design efficient and specific inhibitors in addition to those already used in therapy that are not highly specific and are responsible for the side effects exerted by clinical approach to either Parkinson's disease or aromatic amino acid decarboxylase deficiency. PMID:24407024

  13. Effect of inhibitors of arachidonic acid metabolism on alpha-aminoisobutyric acid transport in human lymphocytes.

    PubMed

    Udey, M C; Parker, C W

    1982-02-01

    The role of arachidonic acid metabolism (or metabolites) in the modulation of alpha-aminoisobutyric acid transport in resting and concanavalin A-stimulated human peripheral blood lymphocytes was evaluated using previously characterized inhibitors of arachidonic acid metabolism. Nordihydroguairetic acid (a nonselective antioxidant), 5,8,11,14-eicosatetraynoic acid (an inhibitor of lipoxygenase and cyclooxygenase activities), indomethacin and acetylsalicylic acid (selective cyclooxygenase inhibitors), and 1-benzylimidazole, Ro-22-3581 and Ro-22-3582 (thromboxane synthetase inhibitors) proved to be potent inhibitors of amino acid transport activity in normal resting and lectin-activated lymphocytes at concentrations known to decrease thromboxane A2 production. The rank order of effectiveness of these various inhibitors compared favorably with their relative potencies as inhibitors of thromboxane B2 synthesis under the same conditions, as determined by radioimmunoassay. Inhibitory effects noted were not due to overt cytotoxicity and seemed to involve changes primarily in the Vmax and not the Km of the transport process. Drug-induced alterations in the magnitude of concanavalin A binding were not observed. These results suggest that the activity of amino acid transport systems can be influenced by certain arachidonic acid metabolites, probably thromboxanes, in both stimulated and unstimulated lymphocytes. In addition, these findings may provide a partial explanation for the observation that inhibitors of thromboxane formation prevent lymphocyte mitogenesis.

  14. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

    PubMed

    Shi, Feng; Jiang, Junjun; Li, Yongfu; Li, Youxin; Xie, Yilong

    2013-11-01

    γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

  15. In vitro inhibition of lysine decarboxylase activity by organophosphate esters.

    PubMed

    Wang, Sufang; Wan, Bin; Zhang, Lianying; Yang, Yu; Guo, Liang-Hong

    2014-12-01

    Organophosphate esters (OPEs), a major group of organophosphorus flame retardants, are regarded as emerging environmental contaminants of health concern. Amino acid decarboxylases catalyze the conversion of amino acids into polyamines that are essential for cell proliferation, hypertrophy and tissue growth. In this paper, inhibitory effect of twelve OPEs with aromatic, alkyl or chlorinated alkyl substituents on the activity of lysine decarboxylase (LDC) was assessed quantitatively with an economic and label-free fluorescence sensor and cell assay. The sensor comprises a macrocyclic host (cucurbit[7]uril) and a fluorescent dye (acridine orange) reporter. The twelve OPEs were found to vary in their capacity to inhibit LDC activity. Alkyl group substituted OPEs had no inhibitory effect. By contrast, six OPEs substituted with aromatic or chlorinated alkyl groups inhibited LDC activity significantly with IC50 ranging from 1.32 μM to 9.07 μM. Among them, the inhibitory effect of tri-m-cresyl phosphate (TCrP) was even more effective as an inhibitor than guanosine 5'-diphosphate-3'-diphosphate (ppGpp) (1.60 μM), an LDC natural inhibitor in vivo. Moreover, at non-cytotoxic concentrations, these six OPEs showed perceptible inhibitory effects on LDC activity in PC12 living cells, and led to a marked loss in the cadaverine content. Molecular docking analysis of the LDC/OPE complexes revealed that different binding modes contribute to the difference in their inhibitory effect. Our finding suggested that LDC, as a new potential biological target of OPEs, might be implicated in toxicological and pathogenic mechanism of OPEs. PMID:25264276

  16. Converting maslinic acid into an effective inhibitor of acylcholinesterases.

    PubMed

    Schwarz, Stefan; Loesche, Anne; Lucas, Susana Dias; Sommerwerk, Sven; Serbian, Immo; Siewert, Bianka; Pianowski, Elke; Csuk, René

    2015-10-20

    During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related derivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholinesterases, up to now maslinic acid has not been part of such studies. For this reason, three series of maslinic acid derivatives possessing modifications at different centers were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants comparable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore, additional selectivity as well as cytotoxicity studies were performed underlining the potential of several derivatives and qualifying them for further investigations. Docking studies revealed that the different kinetic behavior within the same compound series may be explained by the ability of the compounds to enter the active site gorge of AChE. PMID:26383128

  17. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  18. Tomato Glutamate Decarboxylase Genes SlGAD2 and SlGAD3 Play Key Roles in Regulating γ-Aminobutyric Acid Levels in Tomato (Solanum lycopersicum).

    PubMed

    Takayama, Mariko; Koike, Satoshi; Kusano, Miyako; Matsukura, Chiaki; Saito, Kazuki; Ariizumi, Tohru; Ezura, Hiroshi

    2015-08-01

    Tomato (Solanum lycopersicum) can accumulate relatively high levels of γ-aminobutyric acid (GABA) during fruit development. However, the molecular mechanism underlying GABA accumulation and its physiological function in tomato fruits remain elusive. We previously identified three tomato genes (SlGAD1, SlGAD2 and SlGAD3) encoding glutamate decarboxylase (GAD), likely the key enzyme for GABA biosynthesis in tomato fruits. In this study, we generated transgenic tomato plants in which each SlGAD was suppressed and those in which all three SlGADs were simultaneously suppressed. A significant decrease in GABA levels, i.e. 50-81% compared with wild-type (WT) levels, was observed in mature green (MG) fruits of the SlGAD2-suppressed lines, while a more drastic reduction (up to <10% of WT levels) was observed in the SlGAD3- and triple SlGAD-suppressed lines. These findings suggest that both SlGAD2 and SlGAD3 expression are crucial for GABA biosynthesis in tomato fruits. The importance of SlGAD3 expression was also confirmed by generating transgenic tomato plants that over-expressed SlGAD3. The MG and red fruits of the over-expressing transgenic lines contained higher levels of GABA (2.7- to 5.2-fold) than those of the WT. We also determined that strong down-regulation of the SlGADs had little effect on overall plant growth, fruit development or primary fruit metabolism under normal growth conditions.

  19. A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells.

    PubMed

    Knight, R R; Dolton, G; Kronenberg-Versteeg, D; Eichmann, M; Zhao, M; Huang, G C; Beck, K; Cole, D K; Sewell, A K; Skowera, A; Peakman, M

    2015-01-01

    CD8 T cells specific for islet autoantigens are major effectors of β cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114-123 (VMNILLQYVV), with studies demonstrating both 114-123 and 114-122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) β cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114-122 alone or both 114-122 and 114-123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114-123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201(+) human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114-123 as the predominant epitope in this region. These studies highlight the importance of understanding β cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells.

  20. Harmonization of Glutamic Acid Decarboxylase and Islet Antigen-2 Autoantibody Assays for National Institute of Diabetes and Digestive and Kidney Diseases Consortia

    PubMed Central

    Bonifacio, Ezio; Yu, Liping; Williams, Alastair K.; Eisenbarth, George S.; Bingley, Polly J.; Marcovina, Santica M.; Adler, Kerstin; Ziegler, Anette G.; Mueller, Patricia W.; Schatz, Desmond A.; Krischer, Jeffrey P.; Steffes, Michael W.; Akolkar, Beena

    2010-01-01

    Background/Rationale: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. Methods: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8–493 World Health Organization (WHO) units/ml and IA-2A 2–235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using 35S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. Results: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). Conclusion: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements. PMID:20444913

  1. The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency.

    PubMed

    Montioli, Riccardo; Paiardini, Alessandro; Kurian, Manju A; Dindo, Mirco; Rossignoli, Giada; Heales, Simon J R; Pope, Simon; Voltattorni, Carla Borri; Bertoldi, Mariarita

    2016-06-01

    We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328-339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed.

  2. Identification of sulfonic acids as efficient ecto-5'-nucleotidase inhibitors.

    PubMed

    Iqbal, Jamshed; Saeed, Aamer; Raza, Rabia; Matin, Abdul; Hameed, Abdul; Furtmann, Norbert; Lecka, Joanna; Sévigny, Jean; Bajorath, Jürgen

    2013-01-01

    Ecto-5'-nucleotidase (CD73) is well known for its implication in cancer. Inhibition of ecto-5'-nucleotidases is thought to provide an attractive approach to cancer therapy. This study identifies sulfonic acid compounds as efficient inhibitors of ecto-5'-nucleotidases. The compounds were tested against recombinant human and rat ecto-5'-nucleotidases. The most potent new sulfonic acid inhibitor 6-amino-4-hydroxynaphthalene-2-sulfonic acid (1) of ecto-5'-nucleotidase had an IC₅₀ of 1.32 ± 0.09 μM for the human and 10.4 ± 3.3 μM for the rat enzyme. Generally, all compounds were more active against the human enzyme. Plausible binding mode models were developed for this new class of inhibitors. Furthermore, several sulfonic acid inhibitors were efficient cytotoxic agents when tested on H157 cancer cell lines. Hence, new ecto-5'-nucleotidases inhibitors displayed significant potential for further development as compounds for anti-cancer therapy.

  3. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    SciTech Connect

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A.

    2010-08-26

    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  4. T-cell reactivity to glutamic acid decarboxylase in stiff-man syndrome and cerebellar ataxia associated with polyendocrine autoimmunity

    PubMed Central

    Costa, M; Saiz, A; Casamitjana, R; Castañer, M FernÁndez; SanmartÍ, A; Graus, F; Jaraquemada, D

    2002-01-01

    Antibodies to glutamic acid decarboxilase (GAD-Abs) are present in the serum of 60–80% of newly diagnosed type 1 diabetes (DM1) patients and patients with autoimmune polyendocrine syndrome (APS) associated with DM1. Higher titre of GAD-Abs are also present in the serum of 60% of patients with stiff-man syndrome (SMS) and all reported patients with cerebellar ataxia associated with polyendocrine autoimmunity (CAPA). Several studies suggest that GAD-Abs may play a critical role in the pathogenesis of SMS and CAPA but little is known about T-cell responsiveness to GAD-65 in these neurological diseases. To analyse cell-mediated responses to GAD, we studied the peripheral blood lymphocyte proliferation and cytokine responses to recombinant human GAD-65 in 5 patients with SMS, 6 with CAPA, 9 with DM1, 8 with APS and 15 control subjects. GAD-65-specific cellular proliferation was significantly higher in SMS than in CAPA, DM1, APS or controls. In contrast, only T cells from CAPA patients showed a significantly high production of interferon-γ after GAD stimulation, compared to all other patients and controls. No differences were found for IL-4 production. These results suggest that, despite similar humoral autoreactivity, cellular responses to GAD are different between SMS and CAPA, with a greater inflammatory response in CAPA, and this difference may be relevant to the pathogenesis of these diseases. PMID:12197888

  5. Cysteinesulfinate decarboxylase: Characterization, inhibition, and metabolic role in taurine formation

    SciTech Connect

    Weinstein, C.L.

    1988-01-01

    Cysteinesulfinate decarboxylase, an enzyme that plays a major role in the formation of taurine from cysteine, has been purified from rat liver to homogeneity and characterized. The physical properties of the enzyme were studied, along with its substrate specificity. Multiple forms of the enzyme were found in rat liver, kidney, and brain with isoelectric points ranging from pH 5.6 to 4.9. These multiple forms did not differ in their substrate specificity. It was found by using gel electrofocusing and polyclonal antibodies raised to the liver enzyme that the different forms of cysteinesulfinate decarboxylase are identical in the various rat tissues studied. Various inhibitors of the enzyme were tested both in vitro and in vivo in order to evaluate the role of cysteinesulfinate decarboxylase in taurine formation in mammalian tissues. In in vitro studies, cysteinesulfinate decarboxylase was irreversibly inhibited by {beta}-ethylidene-DL-aspartate (Ki = 10 mM), and competitive inhibition was found using mercaptomethylsuccinate (Ki = 0.1 mM) and D-cysteinesulfinate (Ki = 0.32 mM) when L-cysteinesulfinate was used as a substrate. In order to be able to test these inhibitors in vivo, L-(1-{sup 14}C)cysteinesulfonate was evaluated as a probe for the in vivo measurement of cysteinesulfinate decarboxylase activity. The metabolism of cysteinesulfonate and the product of its transamination, {beta}-sulfopyruvate, was studied, and it was found that L-(1-{sup 14}C)cysteinesulfonate is an accurate and convenient probe for cysteinesulfinate decarboxylase activity. Using L-(1-{sup 14}C)cysteinesulfonate, it was found that D-cysteinesulfinate inhibits cysteinesulfinate decarboxylase activity by greater than 90% in the intact mouse and that inhibition lasts for up to fifteen hours.

  6. Utilization of Putrescine in Tobacco Cell Lines Resistant to Inhibitors of Polyamine Synthesis 1

    PubMed Central

    Hiatt, Andrew; Malmberg, Russell L.

    1988-01-01

    Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium. Images Fig. 1 PMID:16665927

  7. Polyamines are Inhibitors of Gastric Acid Secretion

    NASA Astrophysics Data System (ADS)

    Ray, Tushar K.; Nandi, Jyotirmoy; Pidhorodeckyj, Nykolai; Meng-Ai, Zhou

    1982-03-01

    The naturally occurring organic polycations such as spermine and spermidine inhibit histamine-stimulated gastric acid secretion by bullfrog gastric mucosa in vitro; spermine is much more potent than spermidine. Unlike the H2 receptor antagonists, the polyamines are completely ineffective from the nutrient side and are effective only from the secretory side of the chambered mucosa. The polyamine effects could be reversed by increasing K+ concentration in the secretory solution. Studies with isolated gastric microsomal vesicles demonstrate that the polyamines do not inhibit the gastric H+,K+-ATPase but greatly decrease the ATPase-mediated uptake of H+ under appropriate conditions. For the latter effects the presence of polyamine within the vesicle interior was found to be essential. Our data strongly suggest an uncoupling of the gastric H+,K+-ATPase system by the polyamines. The therapeutic potential of these and similar compounds in the treatment of hyperacidity and peptic ulcer is discussed.

  8. Design and synthesis of boronic acid inhibitors of endothelial lipase.

    PubMed

    O'Connell, Daniel P; LeBlanc, Daniel F; Cromley, Debra; Billheimer, Jeffrey; Rader, Daniel J; Bachovchin, William W

    2012-02-01

    Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL. PMID:22225633

  9. Discovery of potent wall teichoic acid early stage inhibitors.

    PubMed

    Labroli, Marc A; Caldwell, John P; Yang, Christine; Lee, Sang Ho; Wang, Hao; Koseoglu, Sandra; Mann, Paul; Yang, Shu-Wei; Xiao, Jing; Garlisi, Charles G; Tan, Christopher; Roemer, Terry; Su, Jing

    2016-08-15

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with β-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant Staphylococci. PMID:27436582

  10. Tanzawaic Acids, a Chemically Novel Set of Bacterial Conjugation Inhibitors

    PubMed Central

    Getino, María; Fernández-López, Raúl; Palencia-Gándara, Carolina; Campos-Gómez, Javier; Sánchez-López, Jose M.; Martínez, Marta; Fernández, Antonio; de la Cruz, Fernando

    2016-01-01

    Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination. PMID:26812051

  11. Salicylic Acid Inhibits Synthesis of Proteinase Inhibitors in Tomato Leaves Induced by Systemin and Jasmonic Acid.

    PubMed Central

    Doares, S. H.; Narvaez-Vasquez, J.; Conconi, A.; Ryan, C. A.

    1995-01-01

    Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol Mol Plant Pathol 33: 377-384), and linolenic acid (H. Pena-Cortes, T. Albrecht, S. Prat, E.W. Weiler, L. Willmitzer [1993] Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step in the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes. PMID:12228577

  12. A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site.

    PubMed

    Gallego, P P; Whotton, L; Picton, S; Grierson, D; Gray, J E

    1995-03-01

    A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product gamma-aminobutyric acid (GABA) in fruit, are discussed.

  13. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    PubMed

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

  14. Behavior of aspartic acid as a corrosion inhibitor for steel

    SciTech Connect

    Kalota, D.J.; Silverman, D.C. )

    1994-02-01

    Corrosion inhibition of steel by aspartic acid (C[sub 4]H[sub 7]NO[sub 4]), an amino acid of low molecular weight, was found to depend strongly on pH. At a pH less than the ionization constant at [approximately]9.5 to 10 (measured at 25 C), C[sub 4]H[sub 7]NO[sub 4] appeared to accelerate corrosion. Above the pH, it acted as a corrosion inhibitor for steel. A specially constructed potential-pH diagram for iron (Fe) that incorporated C[sub 4]H[sub 7]NO[sub 4] showed the change in behavior was accompanied by the most stable thermodynamic state changing from an iron aspartate complex to iron oxide. Polymerized C[sub 4]H[sub 7]NO[sub 4] (polyaspartic acid) behaved in a similar manner. Some other amino acids of low molecular weight behaved similarly.

  15. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  16. Thermal copoly/amino acids/ as inhibitors of glyoxalase I

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Syren, R. M.; Windsor, C. R.

    1979-01-01

    A number of copoly(alpha-amino acids) have been prepared thermally; some have been found to function as inhibitors of glyoxalase I, an enzyme which occupies a central position in Szent-Gyorgyi's theory of tumour genesis. These polymers are also of interest in the search for synthetic peptides having carcinostatic activity, since many natural peptides are active. The way in which the inhibitory activity varies with composition of the synthetic polymers has been investigated. Various properties (hydrophobicity, molecular weight, UV absorption, kinetic type) have been examined in a search for correlates of inhibitory activity.

  17. Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis.

    PubMed Central

    Bastola, D. R.; Minocha, S. C.

    1995-01-01

    Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells. PMID:12228581

  18. Uncovering the Lactobacillus plantarum WCFS1 gallate decarboxylase involved in tannin degradation.

    PubMed

    Jiménez, Natalia; Curiel, José Antonio; Reverón, Inés; de Las Rivas, Blanca; Muñoz, Rosario

    2013-07-01

    Lactobacillus plantarum is a lactic acid bacterium able to degrade tannins by the subsequent action of tannase and gallate decarboxylase enzymes. The gene encoding tannase had previously been identified, whereas the gene encoding gallate decarboxylase is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gallic-acid induced L. plantarum extracts showed a 54-kDa protein which was absent in the uninduced cells. This protein was identified as Lp_2945, putatively annotated UbiD. Homology searches identified ubiD-like genes located within three-gene operons which encoded the three subunits of nonoxidative aromatic acid decarboxylases. L. plantarum is the only bacterium in which the lpdC (lp_2945) gene and the lpdB and lpdD (lp_0271 and lp_0272) genes are separated in the chromosome. Combination of extracts from recombinant Escherichia coli cells expressing the lpdB, lpdC, and lpdC genes demonstrated that LpdC is the only protein required to yield gallate decarboxylase activity. However, the disruption of these genes in L. plantarum revealed that the lpdB and lpdC gene products are essential for gallate decarboxylase activity. Similar to L. plantarum tannase, which exhibited activity only in esters derived from gallic and protocatechuic acids, purified His6-LpdC protein from E. coli showed decarboxylase activity against gallic and protocatechuic acids. In contrast to the tannase activity, gallate decarboxylase activity is widely present among lactic acid bacteria. This study constitutes the first genetic characterization of a gallate decarboxylase enzyme and provides new insights into the role of the different subunits of bacterial nonoxidative aromatic acid decarboxylases.

  19. Uncovering the Lactobacillus plantarum WCFS1 Gallate Decarboxylase Involved in Tannin Degradation

    PubMed Central

    Jiménez, Natalia; Curiel, José Antonio; Reverón, Inés; de las Rivas, Blanca

    2013-01-01

    Lactobacillus plantarum is a lactic acid bacterium able to degrade tannins by the subsequent action of tannase and gallate decarboxylase enzymes. The gene encoding tannase had previously been identified, whereas the gene encoding gallate decarboxylase is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gallic-acid induced L. plantarum extracts showed a 54-kDa protein which was absent in the uninduced cells. This protein was identified as Lp_2945, putatively annotated UbiD. Homology searches identified ubiD-like genes located within three-gene operons which encoded the three subunits of nonoxidative aromatic acid decarboxylases. L. plantarum is the only bacterium in which the lpdC (lp_2945) gene and the lpdB and lpdD (lp_0271 and lp_0272) genes are separated in the chromosome. Combination of extracts from recombinant Escherichia coli cells expressing the lpdB, lpdC, and lpdC genes demonstrated that LpdC is the only protein required to yield gallate decarboxylase activity. However, the disruption of these genes in L. plantarum revealed that the lpdB and lpdC gene products are essential for gallate decarboxylase activity. Similar to L. plantarum tannase, which exhibited activity only in esters derived from gallic and protocatechuic acids, purified His6-LpdC protein from E. coli showed decarboxylase activity against gallic and protocatechuic acids. In contrast to the tannase activity, gallate decarboxylase activity is widely present among lactic acid bacteria. This study constitutes the first genetic characterization of a gallate decarboxylase enzyme and provides new insights into the role of the different subunits of bacterial nonoxidative aromatic acid decarboxylases. PMID:23645198

  20. Glycine decarboxylase in Rhodopseudomonas spheroides and in rat liver mitochondria

    PubMed Central

    Tait, G. H.

    1970-01-01

    1. Glycine decarboxylase and glycine–bicarbonate exchange activities were detected in extracts of Rhodopseudomonas spheroides and in rat liver mitochondria and their properties were studied. 2. The glycine decarboxylase activity from both sources is stimulated when glyoxylate is added to the assay system. 3. Several proteins participate in these reactions and a heat-stable low-molecular-weight protein was purified from both sources. 4. These enzyme activities increase markedly when R. spheroides is grown in the presence of glycine, glyoxylate, glycollate, oxalate or serine. 5. All the enzymes required to catalyse the conversion of glycine into acetyl-CoA via serine and pyruvate were detected in extracts of R. spheroides; of these glycine decarboxylase has the lowest activity. 6. The increase in the activity of glycine decarboxylase on illumination of R. spheroides in a medium containing glycine, and the greater increase when ATP is also present in the medium, probably accounts for the increased incorporation of the methylene carbon atom of glycine into fatty acids found previously under these conditions (Gajdos, Gajdos-Török, Gorchein, Neuberger & Tait, 1968). 7. The results are compared with those obtained by other workers on the glycine decarboxylase and glycine–bicarbonate exchange activities in other systems. PMID:5476725

  1. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Galston, A. W.

    1986-01-01

    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  2. cis-Cinnamic acid selective suppressors distinct from auxin inhibitors.

    PubMed

    Okuda, Katsuhiro; Nishikawa, Keisuke; Fukuda, Hiroshi; Fujii, Yoshiharu; Shindo, Mitsuru

    2014-01-01

    The activity of cis-cinnamic acid (cis-CA), one of the allelochemicals, in plants is very similar to that of indole-3-acetic acid (IAA), a natural auxin, and thus cis-CA has long been believed to be an analog of auxin. We have reported some structure-activity relationships studies by synthesizing over 250 cis-CA derivatives and estimating their inhibitory activities on root growth inhibition in lettuce. In this study, the compounds that showed low- or no-activity on root growth inhibition were recruited as candidates suppressors against cis-CA and/or auxin and tested for their activity. In the presence of cis-CA, lettuce root growth was inhibited; however, the addition of some cis-CA derivatives restored control-level root growth. Four compounds, (Z)-3-(4-isopropylphenyl)acrylic acid, (Z)-3-(3-butoxyphenyl)acrylic acid, (Z)-3-[3-(pentyloxy)phenyl]acrylic acid, and (Z)-3-(naphthalen-1-yl)acrylic acid were selected as candidates for a cis-CA selective suppressor they allowed the recovery of root growth from inhibition by cis-CA treatment without any effects on the IAA-induced effect or elongating activity by themselves. Three candidates significantly ameliorated the root shortening by the potent inhibitor derived from cis-CA. In brief, we have found some cis-CA selective suppressors which have never been reported from inactive cis-CA derivatives for root growth inhibition. cis-CA selective suppressors will play an important role in elucidating the mechanism of plant growth regulation.

  3. Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

    PubMed

    Kuo, D J; Jordan, F

    1983-08-01

    (E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

  4. Identification of Bidentate Salicylic Acid Inhibitors of PTP1B

    PubMed Central

    2015-01-01

    PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth in vitro and in vivo. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor 6, identifying new and more potent bidentate PTP1B inhibitors, such as 20h, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, 20f, 20h, and 20j suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, 20f, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, 20i and 20j displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation. PMID:26396684

  5. Study on polyepoxysuccinic acid reverse osmosis scale inhibitor.

    PubMed

    Sun, Yonghong; Xiang, Wenhua; Wang, Ying

    2009-01-01

    The effectiveness and applicability of polyepoxysuccinic acid (PESA) as a reverse osmosis (RO) scale inhibitor were studied using static and dynamic methods. In the static study, PESA performed very well in the treatment of water samples with various types of scale that commonly exist in RO water (i.e., CaCO3, CaSO4, BaSO4, SrSO4), showing an average scale inhibition ratio of above 90% at a dosage of 10 mg/L. SEM analysis showed a disordered CaCO3 crystal in the presence of PESA, suggesting the scale-inhibiting mechanism. In the dynamic experiment, PESA also showed great performance in the treatment of 10 samples of synthetic tap water or sea water with different ion composition (i.e., Ca(2+), HCO3(-), Fe(3+), Al(3+)). After one cycle (7 d) of RO treatment of each sample at full recycle operation mode under a constant pressure of 1×10(6) Pa, the differences in water permeability and desalination ratio among samples are within 5%, suggesting that PESA is capable of treating waters of different quality, and has a strong resistance to ions that are harmful to scale inhibiting (i.e., Fe(3+) and Al(3+)). All these results suggest that PESA should be an effective scale inhibitor that is applicable to RO treatment of waters with a wide range of ion compositions.

  6. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  7. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

    PubMed Central

    Richardson, Graham; Ding, Haizhen; Rocheleau, Tom; Mayhew, George; Reddy, Erin; Han, Qian; Christensen, Bruce M.; Li, Jianyong

    2010-01-01

    A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues. PMID:19842059

  8. Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis1

    PubMed Central

    Birecka, Helena; Garraway, Michael O.; Baumann, Russell J.; McCann, Peter P.

    1986-01-01

    α-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl2. α-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus. PMID:16664707

  9. Three Distinct Glutamate Decarboxylase Genes in Vertebrates

    PubMed Central

    Grone, Brian P.; Maruska, Karen P.

    2016-01-01

    Gamma-aminobutyric acid (GABA) is a widely conserved signaling molecule that in animals has been adapted as a neurotransmitter. GABA is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). Two vertebrate genes, GAD1 and GAD2, encode distinct GAD proteins: GAD67 and GAD65, respectively. We have identified a third vertebrate GAD gene, GAD3. This gene is conserved in fishes as well as tetrapods. We analyzed protein sequence, gene structure, synteny, and phylogenetics to identify GAD3 as a homolog of GAD1 and GAD2. Interestingly, we found that GAD3 was lost in the hominid lineage. Because of the importance of GABA as a neurotransmitter, GAD3 may play important roles in vertebrate nervous systems. PMID:27461130

  10. Boronic acid-based enzyme inhibitors: a review of recent progress.

    PubMed

    Fu, H; Fang, H; Sun, Jie; Wang, H; Liu, A; Sun, J; Wu, Z

    2014-01-01

    Since Bortezomib was approved by US FDA as the first drug to treat multiple myeloma, various boronic acid compounds have been developed as enzyme inhibitors. This paper reviewed the progress of boronic acid-based inhibitors against enzymes including proteasome, serine protease, HDACs and other enzymes in the past decade.

  11. Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: a structural basis for the decarboxylase and hydratase activities.

    PubMed

    Almrud, Jeffrey J; Poelarends, Gerrit J; Johnson, William H; Serrano, Hector; Hackert, Marvin L; Whitman, Christian P

    2005-11-15

    Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.

  12. Terreic Acid, a Quinone Epoxide Inhibitor of Bruton's Tyrosine Kinase

    NASA Astrophysics Data System (ADS)

    Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

    1999-03-01

    Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

  13. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots.

    PubMed

    Hao, Yu-Jin; Kitashiba, Hiroyasu; Honda, Chikako; Nada, Kazuyoshi; Moriguchi, Takaya

    2005-04-01

    Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are two important enzymes responsible for putrescine biosynthesis. In this study, a full-length ADC cDNA (MdADC) was isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Meanwhile, a partial ODC (pMdODC) could be amplified only by a second RCR from the RT-PCR products, whereas a full-length ODC could not be obtained by either cDNA library screening or 5'- and 3'-RACEs, suggesting quite low expression. Moreover, D-arginine, an ADC inhibitor, caused a decrease in ADC activity and severely inhibited the growth of apple callus, which could be partially resumed by exogenous addition of putrescine, whereas alpha-difluoromethylornithine (DFMO), an inhibitor for ODC, caused the incomplete repression of callus growth without changing ODC activity. RNA gel blot showed that the expression level of MdADC was high in young tissues/organs with rapid cell division and was positively induced by chilling, salt, and dehydration, implying its involvement in both cell growth and these stress responses. By contrast, the transcript of ODC could not be detected by RNA gel blot analysis. Based on the present study, it is possible to conclude that (i) the ODC pathway is active in apple, although the expression level of the pMdODC gene homologous with its counterparts found in other plant species is quite low; and (ii) MdADC expression correlates with cell growth and stress responses to chilling, salt, and dehydration, suggesting that ADC is a primary biosynthetic pathway for putrescine biosynthesis in apple.

  14. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

    PubMed Central

    Valdes-Rodriguez, S; Segura-Nieto, M; Chagolla-Lopez, A; Verver y Vargas-Cortina, A; Martinez-Gallardo, N; Blanco-Labra, A

    1993-01-01

    A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima. PMID:8290633

  15. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    PubMed

    Ha, Byung Hak; Cho, Ki Joon; Choi, Yu Jin; Park, Ky Young; Kim, Kyung Hyun

    2004-04-01

    Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.

  16. Detection and transfer of the glutamate decarboxylase gene in Streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GABA (gamma-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermen...

  17. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GABA ('-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented da...

  18. Azorhodanine derivatives as inhibitors for acidic corrosion of nickel.

    PubMed

    Fouda, Abd El-Aziz S; Al-Sarawy, Ahmed A; Omar, Tark M

    2005-01-01

    Azorhodanine derivatives (HL1-HL5) were tested as corrosion inhibitors for nickel in 2M HNO3 solution using weight loss and galvanostatic polarization techniques. The results showed that these derivatives act as inhibitors for nickel in this medium. The inhibition was assumed to occur via adsorption of the inhibitor molecule on the metal surface. Polarization measurements indicated that these compounds act as mixed-type inhibitors, but the cathode is more polarized when an external current was applied. This means that these compounds retard the rate of hydrogen evolution and the rate of dissolution of the metal. Results showed that azorhodanine derivatives are adsorbed on the nickel surface following Temkin's adsorption isotherm. The activation energy and thermodynamic parameters were calculated and discussed at different temperatures (30-45 degrees C).

  19. Development of imidazole alkanoic acids as mGAT3 selective GABA uptake inhibitors.

    PubMed

    Hack, Silke; Wörlein, Babette; Höfner, Georg; Pabel, Jörg; Wanner, Klaus T

    2011-05-01

    A new series of potential GABA uptake inhibitors starting from of 1H-imidazol-4-ylacetic acid with the carboxylic acid side chain originating from different positions and varying in length have been synthesized and tested for the inhibitory potency at the four GABA uptake transporters mGAT1-4 stably expressed in HEK cells. Further two bicyclic compounds with a rigidified carboxylic acid side chain were included in this study. The results of the biological tests indicated that most ω-imidazole alkanoic and alkenoic acid derivatives exhibit the highest potencies as GABA uptake inhibitors at mGAT3.

  20. Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase.

    PubMed

    Mohd-Pahmi, Siti Hajar; Hussein, Waleed M; Schenk, Gerhard; McGeary, Ross P

    2011-05-15

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl derivatives of α-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K(ic)=5 μM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K(ic)=8 μM, the most potent inhibitor of a mammalian PAP yet reported.

  1. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    PubMed

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D

    2010-12-29

    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  2. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    PubMed

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I

    2007-05-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  3. 2-Aminoimidazole Amino Acids as Inhibitors of the Binuclear Manganese Metalloenzyme Human Arginase I

    SciTech Connect

    Ilies, M.; Di Costanzo, L; North, M; Scott, J; Christianson, D

    2010-01-01

    Arginase, a key metalloenzyme of the urea cycle that converts L-arginine into L-ornithine and urea, is presently considered a pharmaceutical target for the management of diseases associated with aberrant L-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the 2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic acid (A1P, 10), binds to human arginase I with K{sub d} = 2 {micro}M and significantly attenuates airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with promising pharmacological profiles.

  4. Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

    DOEpatents

    Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

    2008-02-05

    The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

  5. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants.

    PubMed

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors' therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic-pituitary-adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  6. Synthesis and antibacterial evaluation of anziaic acid and analogues as topoisomerase I inhibitors

    PubMed Central

    Lin, Hao; Annamalai, Thirunavukkarasu; Bansod, Priyanka; Tse-Dinh, Yuk-Ching

    2013-01-01

    Naturally occurring anziaic acid was very recently reported as a topoisomerase I inhibitor with antibacterial activity. Herein total synthesis of anziaic acid and structural analogues is described and the preliminary structure-activity relationship (SAR) has been developed based on topoisomerase inhibition and whole cell antibacterial activity. PMID:24363888

  7. 4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Zhi, Ying; Gao, Li-Xin; Jin, Yi; Tang, Chun-Lan; Li, Jing-Ya; Li, Jia; Long, Ya-Qiu

    2014-07-15

    Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

  8. Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity.

    PubMed

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

    2005-07-01

    Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.

  9. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  10. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  11. Overexpression of Tyrosine hydroxylase and Dopa decarboxylase associated with pupal melanization in Spodoptera exigua

    PubMed Central

    Liu, Sisi; Wang, Mo; Li, Xianchun

    2015-01-01

    Melanism has been found in a wide range of species, but the molecular mechanisms involved remain largely elusive. In this study, we studied the molecular mechanisms of the pupal melanism in Spodoptera exigua. The full length cDNA sequences of tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), two key enzymes in the biosynthesis pathway of melanin, were cloned, and their temporal expression patterns in the integument were compared during the larval-pupal metamorphosis process of the S. exigua wild type (SEW) and melanic mutant (SEM) strains. No amino acid change in the protein sequence of TH and DDC was found between the two strains. Both DDC and TH were significantly over-expressed in the integument of the SEM strain at late-prepupa and 0 h pupa, respectively, compared with those of the SEW strain. Feeding 5th instar larvae of SEM with diets incorporated with 1 mg/g of the DDC inhibitor L-α-Methyl-DOPA and 0.75 mg/g of the TH inhibitor 3-iodo-tyrosine (3-IT) resulted in 20% pupae with partially-rescued phenotype and 68.2% of pupae with partially- or fully-rescued phenotype, respectively. These results indicate that overexpressions of TH and DDC are involved in the pupal melanization of S. exigua. PMID:26084938

  12. Lichen secondary metabolite evernic acid as potential quorum sensing inhibitor against Pseudomonas aeruginosa.

    PubMed

    Gökalsın, Barış; Sesal, Nüzhet Cenk

    2016-09-01

    Cystic Fibrosis is a genetic disease and it affects the respiratory and digestive systems. Pseudomonas aeruginosa infections in Cystic Fibrosis are presented as the main cause for high mortality and morbidity rates. Pseudomonas aeruginosa populations can regulate their virulence gene expressions via the bacterial communication system: quorum sensing. Inhibition of quorum sensing by employing quorum sensing inhibitors can leave the bacteria vulnerable. Therefore, determining natural sources to obtain potential quorum sensing inhibitors is essential. Lichens have ethnobotanical value for their medicinal properties and it is possible that their secondary metabolites have quorum sensing inhibitor properties. This study aims to investigate an alternative treatment approach by utilizing lichen secondary metabolite evernic acid to reduce the expressions of Pseudomonas aeruginosa virulence factors by inhibiting quorum sensing. For this purpose, fluorescent monitor strains were utilized for quorum sensing inhibitor screens and quantitative reverse-transcriptase PCR analyses were conducted for comparison. Results indicate that evernic acid is capable of inhibiting Pseudomonas aeruginosa quorum sensing systems.

  13. Identification of small molecule sulfonic acids as ecto-5'-Nucleotidase inhibitors.

    PubMed

    Raza, Rabia; Saeed, Aamer; Lecka, Joanna; Sévigny, Jean; Iqbal, Jamshed

    2012-11-01

    Ecto-5'-Nucleotidase inhibitors have great potential as anti-tumor agents. We have investigated biochemical properties of human and rat ecto-5'-Nucleotidases and characterized 19 small molecule sulfonic acid derivatives as potential inhibitors of ecto-5'-Nucleotidases. We identified 11 potent inhibitors of human and rat ecto-5'-Nucleotidases and checked their selectivity. Compound 10 (Sodium 2,4-dinitrobenzenesulfonate) with K(i) value of 0.66 μM and 19 (N-(4-sulfamoylphenylcarbamothioyl) pivalamide) with K(i) value of 0.78 μM were identified as the most potent inhibitors for human and rat ecto-5'-Nucleotidase, respectively. The present compounds have low molecular weights, water solubility and equal potency as compared to the reported inhibitors.

  14. Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid.

    PubMed

    Joshi, Amit; Saraph, Arundhati; Poon, Vincent; Mogridge, Jeremy; Kane, Ravi S

    2006-01-01

    We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics.

  15. Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid.

    PubMed

    Joshi, Amit; Saraph, Arundhati; Poon, Vincent; Mogridge, Jeremy; Kane, Ravi S

    2006-01-01

    We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics. PMID:16984137

  16. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    PubMed

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors.

  17. A crystallographic fragment screen identifies cinnamic acid derivatives as starting points for potent Pim-1 inhibitors.

    PubMed

    Schulz, Michèle N; Fanghänel, Jörg; Schäfer, Martina; Badock, Volker; Briem, Hans; Boemer, Ulf; Nguyen, Duy; Husemann, Manfred; Hillig, Roman C

    2011-03-01

    A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.

  18. Inhibitors of fatty acid biosynthesis in sunflower seeds.

    PubMed

    Pleite, Rafael; Martínez-Force, Enrique; Garcés, Rafael

    2006-09-01

    During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds. PMID:16500723

  19. Synthesis and evaluation of new omega-borono-alpha-amino acids as rat liver arginase inhibitors.

    PubMed

    Busnel, Olivier; Carreaux, François; Carboni, Bertrand; Pethe, Stephanie; Goff, Sandrine Vadon-Le; Mansuy, Daniel; Boucher, Jean-Luc

    2005-04-01

    Recent studies have demonstrated that arginase plays important roles in pathologies such as asthma or erectile dysfunctions. We have synthesized new omega-borono-alpha-amino acids that are analogues of the previously known arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) and 2-amino-6-boronohexanoic acid (ABH) and evaluated them as inhibitors of purified rat liver arginase (RLA). In addition to the distance between the B(OH)(2) and the alpha-amino acid functions, the position of the sulfur atom in the side chain also appears as a key determinant for the interaction with the active site of RLA. Furthermore, substitution of the alkyl side chain of BEC by methyl groups and conformational restriction of ABH by incorporation of its side chain in a phenyl ring led to inactive compounds. These results suggest that subtle interactions govern the affinity of inhibitors for the active site of RLA.

  20. Optimization of amino acid thioesters as inhibitors of metallo-β-lactamase L1.

    PubMed

    Liu, Xiao-Long; Yang, Ke-Wu; Zhang, Yue-Juan; Ge, Ying; Xiang, Yang; Chang, Ya-Nan; Oelschlaeger, Peter

    2016-10-01

    The emergence of antibiotic resistance caused by metallo-β-lactamases (MβLs) is a global public health problem. Recently, we found amino acid thioesters to be a highly promising scaffold for inhibitors of the MβL L1. In order to optimize this series of inhibitors, nine new amino acid thioesters were developed by modifying the substituents on the N-terminus of the thioesters and the groups representing the amino acid side chain. Biological activity assays indicate that all of them are very potent inhibitors of L1 with an IC50 value range of 20-600nM, lower than those of most of the previously reported inhibitors of this scaffold. Analysis of structure-activity relationship reveals that big hydrophobic substituents on the N-terminus and a methionine amino acid side chain improves inhibitory activity of the thioesters. All these inhibitors are able to restore antibacterial activity of a β-lactam antibiotic against Escherichia coli BL21(DE3) cells producing L1 to that against E. coli cells lacking a β-lactamase. Docking studies reveal that a large N-terminal hydrophobic group results in a slightly different binding mode than smaller hydrophobic groups at the same position. PMID:27595424

  1. Inhibitors of amino acids biosynthesis as antifungal agents.

    PubMed

    Jastrzębowska, Kamila; Gabriel, Iwona

    2015-02-01

    Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

  2. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.

    PubMed

    Göttlicher, M; Minucci, S; Zhu, P; Krämer, O H; Schimpf, A; Giavara, S; Sleeman, J P; Lo Coco, F; Nervi, C; Pelicci, P G; Heinzel, T

    2001-12-17

    Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. PMID:11742974

  3. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells

    PubMed Central

    Göttlicher, Martin; Minucci, Saverio; Zhu, Ping; Krämer, Oliver H.; Schimpf, Annemarie; Giavara, Sabrina; Sleeman, Jonathan P.; Lo Coco, Francesco; Nervi, Clara; Pelicci, Pier Giuseppe; Heinzel, Thorsten

    2001-01-01

    Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. PMID:11742974

  4. Glutamic Acid Decarboxylase 65 and Islet Cell Antigen 512/IA-2 Autoantibodies in Relation to Human Leukocyte Antigen Class II DR and DQ Alleles and Haplotypes in Type 1 Diabetes Mellitus ▿

    PubMed Central

    Stayoussef, Mouna; Benmansour, Jihen; Al-Jenaidi, Fayza A.; Said, Hichem B.; Rayana, Chiheb B.; Mahjoub, Touhami; Almawi, Wassim Y.

    2011-01-01

    The frequencies of autoantibodies against glutamic acid decarboxylase 65 (GAD65) and islet cell antigen (ICA) 512/IA-2 (512/IA-2) are functions of the specific human leukocyte antigen (HLA) in type 1 diabetes mellitus (T1D). We investigated the association of HLA class II (DR and DQ) alleles and haplotypes with the presence of GAD and IA-2 autoantibodies in T1D. Autoantibodies were tested in 88 Tunisian T1D patients and 112 age- and gender-matched normoglycemic control subjects by enzyme immunoassay. Among T1D patients, mean anti-GAD antibody titers were higher in the DRB1*030101 allele (P < 0.001), together with the DRB1*030101/DQB1*0201 (P < 0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes, while lower anti-GAD titers were associated with the DRB1*070101 (P = 0.001) and DRB1*110101 (P < 0.001) alleles and DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1*030101 (P = 0.001) haplotypes. Mean anti-IA-2 antibody titers were higher in the DRB1*040101 allele (P = 0.007) and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes but were lower in the DRB1*110101 allele (P = 0.010) and the DRB1*110101 (P < 0.001) and DRB1*110101/DQB1*030101 (P = 0.025) haplotypes. Multinomial regression analysis confirmed the positive association of DRB1*030101 and the negative association of DRB1*110101 and DQB1*030101, along with the DRB1*070101/DQB1*0201 and DRB1*110101/DQB1*030101 haplotypes, with anti-GAD levels. In contrast, only the DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-IA-2 titers. Increased GAD65 and IA-2 antibody positivity is differentially associated with select HLA class II alleles and haplotypes, confirming the heterogeneous nature of T1D. PMID:21490167

  5. STEREOLOGICAL ESTIMATES OF THE BASAL FOREBRAIN CELL POPULATION IN THE RAT, INCLUDING NEURONS CONTAINING CHOLINE ACETYLTRANSFERASE (ChAT), GLUTAMIC ACID DECARBOXYLASE (GAD) OR PHOSPHATE-ACTIVATED GLUTAMINASE (PAG) AND COLOCALIZING VESICULAR GLUTAMATE TRANSPORTERS (VGluTs)

    PubMed Central

    GRITTI, I.; HENNY, P.; GALLONI, F.; MAINVILLE, L.; MARIOTTI, M.; JONES, B. E.

    2006-01-01

    The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and are comprised by GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ~355,000 and the numbers of ChAT-immuno-positive (+) as ~22,000, GAD+ ~119,000 and PAG+ ~316,000, corresponding to ~5%, ~35% and ~90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also have the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, VGluT3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or

  6. Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors.

    PubMed

    Varga, Gergely; Docsa, Tibor; Gergely, Pál; Juhász, László; Somsák, László

    2013-03-15

    Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had Ki values of 2.0μM against AMP (competitive) and 3.36μM against glucose-1-phosphate (non-competitive).

  7. Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.

    PubMed

    Wolz, R L; Zeggaf, C; Stöcker, W; Zwilling, R

    1990-09-01

    Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.

  8. Impacts of lignocellulose-derived inhibitors on L-lactic acid fermentation by Rhizopus oryzae.

    PubMed

    Zhang, Li; Li, Xin; Yong, Qiang; Yang, Shang-Tian; Ouyang, Jia; Yu, Shiyuan

    2016-03-01

    Inhibitors generated in the pretreatment and hydrolysis of corn stover and corn cob were identified. In general, they inhibited cell growth, lactate dehydrogenase, and lactic acid production but with less or no adverse effect on alcohol dehydrogenase and ethanol production in batch fermentation by Rhizopus oryzae. Furfural and 5-hydroxymethyl furfural (HMF) were highly toxic at 0.5-1 g L(-1), while formic and acetic acids at less than 4 g L(-1) and levulinic acid at 10 g L(-1) were not toxic. Among the phenolic compounds at 1 g L(-1), trans-cinnamic acid and syringaldehyde had the highest toxicity while syringic, ferulic and p-coumaric acids were not toxic. Although these inhibitors were present at concentrations much lower than their separately identified toxic levels, lactic acid fermentation with the hydrolysates showed much inferior performance compared to the control without inhibitor, suggesting synergistic or compounded effects of the lignocellulose-degraded compounds on inhibiting lactic acid fermentation. PMID:26724548

  9. Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation

    PubMed Central

    Jump, Donald B.; Torres-Gonzalez, Moises; Olson, L. Karl

    2010-01-01

    Acetyl CoA carboxylase (ACC1 & ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL & palmitate (16:0) and linoleate (18:2,n-6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 & 18:2,n-6; IC50 ~ 5 nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2,n-6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids. PMID:21184748

  10. Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design.

    PubMed

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P; Thorn, Katherine J; Christianson, David W

    2011-08-11

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  11. Binding of [alpha, alpha]-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design

    SciTech Connect

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W.

    2011-10-21

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of {alpha},{alpha}-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional {alpha}-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  12. Curcumin Derivatives as Green Corrosion Inhibitors for α-Brass in Nitric Acid Solution

    NASA Astrophysics Data System (ADS)

    Fouda, A. S.; Elattar, K. M.

    2012-11-01

    1,7- Bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-4-arylazo-3,5-dione I-V have been investigated as corrosion inhibitors for α-brass in 2 M nitric acid solution using weight-loss and galvanostatic polarization techniques. The efficiency of the inhibitors increases with the increase in the inhibitor concentration but decreases with a rise in temperature. The conjoint effect of the curcumin derivatives and KSCN has also been studied. The apparent activation energy ( E a*) and other thermodynamic parameters for the corrosion process have also been calculated. The galvanostatic polarization data indicated that the inhibitors were of mixed-type, but the cathode is more polarized than the anode. The slopes of the cathodic and anodic Tafel lines ( b c and b a) are maintained approximately equal for various inhibitor concentrations. However, the value of the Tafel slopes increases together as inhibitor concentration increases. The adsorption of these compounds on α-brass surface has been found to obey the Frumkin's adsorption isotherm. The mechanism of inhibition was discussed in the light of the chemical structure of the undertaken inhibitors.

  13. Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: purification, properties, and amino acid sequences.

    PubMed

    Haldar, U C; Saha, S K; Beavis, R C; Sinha, N K

    1996-02-01

    Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is at pH 4.55 for LA-1 and at pH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 A. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0 x 10(9) M-1 sec-1 for LA-1 and 0.8 x 10(9) M-1 sec-1 for LA-2 and that of K2HPO4 quenching is 1.6 x 10(11) M-1 sec-1 for LA-1 and 1.2 x 10(11) M-1 sec-1 for LA-2. Analysis of the circular dichroic spectra yields 40% alpha-helix and 60% beta-turn for La-1 and 45% alpha-helix and 55% beta-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzyme-inhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors. PMID:8924202

  14. Beyond gastric acid reduction: Proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells

    SciTech Connect

    Becker, Jan C. . E-mail: beckeja@uni-muenster.de; Grosser, Nina; Waltke, Christian; Schulz, Stephanie; Erdmann, Kati; Domschke, Wolfram; Schroeder, Henning; Pohle, Thorsten

    2006-07-07

    Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.

  15. Organic corrosion inhibitors for aluminum in perchloric acid

    SciTech Connect

    Metikos-Hukovic, M.; Stupnisek-Lisac, E. . Dept. of Electrochemistry); Grubac, Z. . Dept. of Inorganic Chemistry)

    1994-02-01

    Substituted N-arylpyrroles containing carbaldehyde groups on a pyrrole ring and their inhibitive effects on the corrosion of aluminum (Al) in perchloric acid (HCIO[sub 4]) were investigated. Electrochemical measurements showed the organic compounds examined had good inhibiting properties at 40 C. The high inhibition effect of the N-aryl-2,5-dimethylpyrroles containing carbaldehyde groups on a pyrrole ring on corrosion of Al in acid media was explained on the basis of the electronic structure of the molecule and by the condensation characteristic of the carbaldehydes. Investigation of the influence of substitution type ([minus]F,[minus]Cl,[minus]NH[sub 2]) on the inhibiting characteristics of examined organic compounds showed the best inhibiting effect was from substituted N-arylpyrrole with fluorine in the ortho position of the phenyl ring.

  16. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

    PubMed

    Gamat, Melissa; Malinowski, Rita L; Parkhurst, Linnea J; Steinke, Laura M; Marker, Paul C

    2015-01-01

    The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating

  17. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate

    PubMed Central

    Gamat, Melissa; Malinowski, Rita L.; Parkhurst, Linnea J.; Steinke, Laura M.; Marker, Paul C.

    2015-01-01

    The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating

  18. HALOACETIC ACIDS AND KINASE INHIBITORS PERTURB MOUSE NEURAL CREST CELLS IN VITRO

    EPA Science Inventory

    HUNTER, E.S.1, J. SMITH2, J. ANDREWS1. 1 Reproductive Toxicology Division, NHEERL, US EPA, Research Triangle Park and 2 Department of Cell and Developmental Biology, UNC-CH, Chapel Hill, North Carolina. Haloacetic acids and kinase inhibitors perturb mouse neural crest cells in vi...

  19. Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Co-application of certain types of compounds with conventional antimicrobial drugs results in the enhancement of efficacy of drugs through a mechanism termed chemosensitization. We show that kojic acid (KA), a natural product, is a potent chemosensitizer to complex III inhibitors of mitochondrial re...

  20. Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells

    PubMed Central

    Winkler, Katrin; Ramer, Robert; Dithmer, Sophie; Ivanov, Igor; Merkord, Jutta; Hinz, Burkhard

    2016-01-01

    Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified. PMID:26930716

  1. Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

    PubMed Central

    Connil, Nathalie; Le Breton, Yoann; Dousset, Xavier; Auffray, Yanick; Rincé, Alain; Prévost, Hervé

    2002-01-01

    Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 μg ml−1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes. PMID:12089039

  2. Retina maturation following administration of thyroxine in developing rats: effects on polyamine metabolism and glutamate decarboxylase.

    PubMed

    Macaione, S; Di Giorgio, R M; Nicotina, P A; Ientile, R

    1984-08-01

    The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and gamma-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9-12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S-Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.

  3. Aerobically incubated medium for decarboxylase testing of Enterobacteriaceae by replica-plating methods.

    PubMed

    Maccani, J E

    1979-12-01

    An aerobically incubated, agar-based medium was developed for amino acid decarboxylase testing of Enterobacteriaceae family members by replica-plating methods. Results with the new medium agreed 97 to 99% with the reference broth method of Moeller, and no false-positive reactions were encountered.

  4. Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation

    SciTech Connect

    Sievers, Stuart A.; Karanicolas, John; Chang, Howard W.; Zhao, Anni; Jiang, Lin; Zirafi, Onofrio; Stevens, Jason T.; Münch, Jan; Baker, David; Eisenberg, David

    2011-09-20

    Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-D-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimer's disease, and a non-natural L-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-{beta}-sheet 'steric zippers', the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.

  5. Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

    PubMed Central

    Min, Xiaoshan; Thibault, Stephen T.; Porter, Amy C.; Gustin, Darin J.; Carlson, Timothy J.; Xu, Haoda; Lindstrom, Michelle; Xu, Guifen; Uyeda, Craig; Ma, Zhihua; Li, Yihong; Kayser, Frank; Walker, Nigel P. C.; Wang, Zhulun

    2011-01-01

    Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition. PMID:21502526

  6. Identification of Anziaic Acid, a Lichen Depside from Hypotrachyna sp., as a New Topoisomerase Poison Inhibitor

    PubMed Central

    Cheng, Bokun; Cao, Shugeng; Vasquez, Victor; Annamalai, Thirunavukkarasu; Tamayo-Castillo, Giselle; Clardy, Jon; Tse-Dinh, Yuk-Ching

    2013-01-01

    Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds. PMID:23593306

  7. Triacontanol negatively modulates the jasmonic acid-stimulated proteinase inhibitors in tomato (Lycopersicon esculentum).

    PubMed

    Ramanarayan, Krishnamurthy; Swamy, Gangadharamurthy Sivakumar

    2004-04-01

    Triacontanol (TRIA), a long chain aliphatic alcohol (C30H61OH) reverses the effect of jasmonic acid (JA) in inducing proteinase inhibitors (PIs) in tomato leaves. Porcine pancreas trypsin and Spodoptera litura gut proteinases were inhibited in the presence of leaf proteins treated with JA, and TRIA partially reverses this effect. Spodoptera litura larvae fed with tomato leaves treated with JA were reduced in body weight and TRIA is able to partially reverse this JA-induced effect. These results reflect the partial reversal effect of TRIA in down regulating the JA-induced production of proteinase inhibitors.

  8. A dual inhibitor of cyclooxygenase and 5-lipoxygenase protects against kainic acid-induced brain injury.

    PubMed

    Minutoli, Letteria; Marini, Herbert; Rinaldi, Mariagrazia; Bitto, Alessandra; Irrera, Natasha; Pizzino, Gabriele; Pallio, Giovanni; Calò, Margherita; Adamo, Elena Bianca; Trichilo, Vincenzo; Interdonato, Monica; Galfo, Federica; Squadrito, Francesco; Altavilla, Domenica

    2015-06-01

    Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity. PMID:25893744

  9. Plant ornithine decarboxylase is not post-transcriptionally feedback regulated by polyamines but can interact with a cytosolic ribosomal protein S15 polypeptide.

    PubMed

    Illingworth, Crista; Michael, Anthony J

    2012-02-01

    The formation of putrescine by ornithine decarboxylase (ODC) is a key regulatory step in polyamine biosynthesis in metazoa and fungi. Excess polyamines post-transcriptionally induce the synthesis of a unique non-competitive protein inhibitor of ODC, termed antizyme. Binding of antizyme to an ODC monomer subunit results in enzymatic inhibition, rapid ubiquitin-independent degradation of ODC by the 26S proteasome and recycling of antizyme. Plants possess an additional route for synthesizing putrescine via arginine decarboxylase (ADC). No homologue of ODC antizyme has been detected in plant genomes but several biochemical studies have reported plant ODC antizyme proteins of 9 and 16 kDa. Here we show that plant cells grown in liquid culture do not exhibit any substantial post-transcriptional, polyamine-responsive feedback regulation of ODC or ADC. However, using the yeast two hybrid system, a plant ODC-binding polypeptide was detected: the C-terminal 84-87 amino acids of cytosolic ribosomal protein (rp) S15. The Arabidopsis rpS15 polypeptide interacted specifically with plant ODC but not with human or Saccharomyces cerevisiae ODCs. Co-expression of either the full length or C-terminal rpS15 polypeptides with a plant ODC in yeast did not reduce ODC enzymatic activity. Only the full length mRNA encoding rpS15 was detected in Arabidopsis cells, suggesting that the C-terminal rpS15 polypeptide is encoded by a low abundance mRNA or the polypeptide is not physiologically relevant in plants. These results confirm the primacy of S-adenosylmethionine decarboxylase as the key regulatory enzyme in plant polyamine biosynthesis. PMID:21814791

  10. Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine

    PubMed Central

    Zhu, Meng-Yang; Iyo, Abiye; Piletz, John E.; Regunathan, Soundar

    2011-01-01

    Agmatine, an amine formed by decarboxylation of L-arginine by arginine decarboxylase (ADC), has been recently discovered in mammalian brain and other tissues. While the cloning and sequencing of ADC from plant and bacteria have been reported extensively, the structure of mammalian enzyme is not known. Using homology screening approach, we have identified a human cDNA clone that exhibits ADC activity when expressed in COS-7 cells. The cDNA and deduced amino acid sequence of this human ADC clone is distinct from ADC of other forms. Human ADC is a 460-amino acid protein that shows about 48% identity to mammalian ornithine decarboxylase (ODC) but has no ODC activity. While naive COS-7 cells do not make agmatine, these cells are able to produce agmatine, as measured by HPLC, when transfected with ADC cDNA. Northern blot analysis using the cDNA probe indicated the expression of ADC message in selective human brain regions and other human tissues. PMID:14738999

  11. Molecular characterization of Mtb-OMP decarboxylase by modeling, docking and dynamic studies.

    PubMed

    Madhusudana, P; Babajan, B; Chaitanya, M; Anuradha, C M; Shobharani, C; Chikati, Rajasekar; Kumar, Chitta Suresh; Rao, K R S Sambasiva; Poda, Sudhakar

    2012-06-01

    Tuberculosis (TB), the second most deadly disease in the world is caused by Mycobacterium tuberculosis (Mtb). In the present work a unique enzyme of Mtb orotidine 5' monophosphate decarboxylase (Mtb-OMP Decase) is selected as drug target due to its indispensible role in biosynthesis of pyrimidines. The present work is focused on understanding the structural and functional aspects of Mtb-OMP Decase at molecular level. Due to absence of crystal structure, the 3D structure of Mtb-OMP Decase was predicted by MODELLER9V7 using a known structural template 3L52. Energy minimization and refinement of the developed 3D model was carried out with Gromacs 3.2.1 and the optimized homology model was validated by PROCHECK,WHAT-IF and PROSA2003. Further, the surface active site amino acids were quantified by WHAT-IF pocket. The exact binding interactions of the ligands, 6-idiouridine 5' monophosphate and its designed analogues with the receptor Mtb-OMP Decase were predicted by docking analysis with AUTODOCK 4.0. This would be helpful in understanding the blockade mechanism of OMP Decase and provide a candidate lead for the discovery of Mtb-OMP Decase inhibitors, which may bring insights into outcome new therapy to treat drug resistant Mtb.

  12. Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity

    PubMed Central

    Realini, Natalia; Solorzano, Carlos; Pagliuca, Chiara; Pizzirani, Daniela; Armirotti, Andrea; Luciani, Rosaria; Costi, Maria Paola; Bandiera, Tiziano; Piomelli, Daniele

    2013-01-01

    The expression of acid ceramidase (AC) – a cysteine amidase that hydrolyses the proapoptotic lipid ceramide – is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents. PMID:23301156

  13. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  14. N-Benzyl-indolo carboxylic acids: Design and synthesis of potent and selective adipocyte fatty-acid binding protein (A-FABP) inhibitors.

    PubMed

    Barf, Tjeerd; Lehmann, Fredrik; Hammer, Kristin; Haile, Saba; Axen, Eva; Medina, Carmen; Uppenberg, Jonas; Svensson, Stefan; Rondahl, Lena; Lundbäck, Thomas

    2009-03-15

    Small molecule inhibitors of adipocyte fatty-acid binding protein (A-FABP) have gained renewed interest following the recent publication of pharmacologically beneficial effects of such inhibitors. Despite the potential utility of selective A-FABP inhibitors within the fields of metabolic disease, inflammation and atherosclerosis, there are few examples of useful A-FABP inhibitors in the public domain. Herein, we describe the optimization of N-benzyl-tetrahydrocarbazole derivatives through the use of co-crystal structure guided medicinal chemistry efforts. This led to the identification of a potent and selective class of A-FABP inhibitors as illustrated by N-benzyl-hexahydrocyclohepta[b]indole 30. PMID:19217286

  15. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

    PubMed

    Shimajiri, Yasuka; Oonishi, Takayuki; Ozaki, Kae; Kainou, Kumiko; Akama, Kazuhito

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.

  16. Lichen secondary metabolite evernic acid as potential quorum sensing inhibitor against Pseudomonas aeruginosa.

    PubMed

    Gökalsın, Barış; Sesal, Nüzhet Cenk

    2016-09-01

    Cystic Fibrosis is a genetic disease and it affects the respiratory and digestive systems. Pseudomonas aeruginosa infections in Cystic Fibrosis are presented as the main cause for high mortality and morbidity rates. Pseudomonas aeruginosa populations can regulate their virulence gene expressions via the bacterial communication system: quorum sensing. Inhibition of quorum sensing by employing quorum sensing inhibitors can leave the bacteria vulnerable. Therefore, determining natural sources to obtain potential quorum sensing inhibitors is essential. Lichens have ethnobotanical value for their medicinal properties and it is possible that their secondary metabolites have quorum sensing inhibitor properties. This study aims to investigate an alternative treatment approach by utilizing lichen secondary metabolite evernic acid to reduce the expressions of Pseudomonas aeruginosa virulence factors by inhibiting quorum sensing. For this purpose, fluorescent monitor strains were utilized for quorum sensing inhibitor screens and quantitative reverse-transcriptase PCR analyses were conducted for comparison. Results indicate that evernic acid is capable of inhibiting Pseudomonas aeruginosa quorum sensing systems. PMID:27465850

  17. 3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis, molecular docking and biological evaluation.

    PubMed

    Shi, Wei-Kang; Deng, Rui-Cheng; Wang, Peng-Fei; Yue, Qin-Qin; Liu, Qi; Ding, Kun-Ling; Yang, Mei-Hui; Zhang, Hong-Yu; Gong, Si-Hua; Deng, Min; Liu, Wen-Run; Feng, Qiu-Ju; Xiao, Zhu-Ping; Zhu, Hai-Liang

    2016-10-01

    Helicobacter pylori urease is involved in several physiologic responses such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids were synthesized and evaluated for urease inhibition. Out of these compounds, 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active inhibitor with IC50 of 0.15±0.05μM, showing a mixed inhibition with both competitive and uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and 0.12μg·mL(-1) for Ki and Ki', respectively. The plasma protein binding assays suggested that d24 exhibited moderate binding to human and rabbit plasma proteins. PMID:27492194

  18. Synthesis and evaluation of aminophosphinic acid derivatives as inhibitors of renal dipeptidase.

    PubMed

    Gurulingappa, Hallur; Buckhalts, Phillip; Kinzler, Kenneth W; Vogelstein, Bert; Khan, Saeed R

    2004-07-01

    Renal dipeptidase (RDP) is an enzyme overexpressed in benign and malignant colorectal tumors. In an effort to identify potent inhibitors of this enzyme, a series of aminophosphinic acid derivatives were synthesized. Compounds 3a and 3c in which the phenyl ring was para substituted with F and Br and olefin with Z geometry, showed better inhibitory activity against RDP enzyme (IC50 = 5-6 nM).

  19. Products of the Black Sea alga Phyllophora nervosa as corrosion inhibitor for steel in acids

    SciTech Connect

    Popelyukh, G.M.; Andrianov, A.M.; Burtnenko, L.M.; Gazha, P.A.; Talavira, L.I.

    1986-05-01

    The authors have investigated the inhibiting properties of the processing products of the Black Sea red seaweed Phyllophora nervosa on specimens of steel St3 in phosphoric and hydrochloric acids of various concentrations at temperatures in the range from 30 to 95 /sup 0/C. They have studied how the concentrations of urotropin, sodium chloride, and Fe/sup 3 +/ ions influence the protective properties of the seaweed inhibitor. They have made preliminary investigations of the mechanisms of the protective action.

  20. Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

    PubMed

    Mielecki, Marcin; Lesyng, Bogdan

    2016-01-01

    Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

  1. How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production.

    PubMed

    Marconi, Patricia L; Alvarez, María A; Pitta-Alvarez, Sandra I

    2007-01-01

    Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhydrazone (MGBG) and the phenylalanine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250- 1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots. PMID:17416978

  2. [Cyclooxygenase inhibitors in some dietary vegetables inhibit platelet aggregation function induced by arachidonic acid].

    PubMed

    Wang, Xin-Hua; Shao, Dong-Hua; Liang, Guo-Wei; Zhang, Ru; Xin, Qin; Zhang, Tao; Cao, Qing-Yun

    2011-10-01

    The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.

  3. The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

    PubMed

    Katane, Masumi; Matsuda, Satsuki; Saitoh, Yasuaki; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2013-08-20

    d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

  4. Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer.

    PubMed

    Melisi, Davide; Caputo, Rosa; Damiano, Vincenzo; Bianco, Roberto; Veneziani, Bianca Maria; Bianco, A Raffaele; De Placido, Sabino; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-12-01

    Biphosphonates (BPs) are widely used to inhibit osteoclastic activity in malignant diseases such as bone metastatic breast and prostate carcinoma. Recent studies reported that BPs could also cause a direct antitumor effect, probably due to their ability to interfere with several intracellular signalling molecules. The enzyme cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) play an important role in the control of cancer cell growth and inhibitors of COX-2 and EGFR have shown antitumor activity in vitro and in vivo in several tumor types. We, and others, have previously shown that EGFR and COX-2 may be directly related to each other and that their selective inhibitors may have a cooperative effect. In the present study we have evaluated the combined effect of zoledronic acid, the most potent nitrogen-containing BP, with the COX-2 inhibitor SC-236 and the selective EGFR-tyrosine kinase inhibitor gefitinib, on breast and prostate cancer models in vitro and in xenografted nude mice. We show that combination of zoledronic acid with SC-236 and gefitinib causes a cooperative antitumor effect accompanied by induction of apoptosis and regulation of the expression of mitogenic factors, proangiogenic factors and cell cycle controllers both in vitro and in xenografted nude mice. The modulatory effect on protein expression and the inhibitory effect on tumor growth is much more potent when the three agents are used together. Since studies are ongoing to explore the antitumor effect of zoledronic acid, our results provide new insights into the mechanism of action of these agents and a novel rationale to translate this feasible combination treatment strategy into a clinical setting.

  5. Discovery of novel, non-acidic mPGES-1 inhibitors by virtual screening with a multistep protocol

    PubMed Central

    Noha, Stefan M.; Fischer, Katrin; Koeberle, Andreas; Garscha, Ulrike; Werz, Oliver; Schuster, Daniela

    2015-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors are considered as potential therapeutic agents for the treatment of inflammatory pain and certain types of cancer. So far, several series of acidic as well as non-acidic inhibitors of mPGES-1 have been discovered. Acidic inhibitors, however, may have issues, such as loss of potency in human whole blood and in vivo, stressing the importance of the design and identification of novel, non-acidic chemical scaffolds of mPGES-1 inhibitors. Using a multistep virtual screening protocol, the Vitas-M compound library (∼1.3 million entries) was filtered and 16 predicted compounds were experimentally evaluated in a biological assay in vitro. This approach yielded two molecules active in the low micromolar range (IC50 values: 4.5 and 3.8 μM, respectively). PMID:26088337

  6. Suppression of asymmetric acid efflux and gravitropism in maize roots treated with auxin transport inhibitors of sodium orthovanadate

    NASA Technical Reports Server (NTRS)

    Mulkey, T. J.; Evans, M. L.

    1982-01-01

    In gravitropically stimulated roots of maize (Zea mays L., hybrid WF9 x 38MS), there is more acid efflux on the rapidly growing upper side than on the slowly growing lower side. In light of the Cholodny/Went hypothesis of gravitropism which states that gravitropic curvature results from lateral redistribution of auxin, the effects of auxin transport inhibitors on the development of acid efflux asymmetry and curvature in gravistimulated roots were examined. All the transport inhibitors tested prevented both gravitropism and the development of asymmetric acid efflux in gravistimulated roots. The results indicate that auxin redistribution may cause the asymmetry of acid efflux, a finding consistent with the Cholodny/Went hypothesis of gravitropism. As further evidence that auxin-induced acid efflux asymmetry may mediate gravitropic curvature, sodium orthovanadate, an inhibitor of auxin-induced H+ efflux was found to prevent both gravitropism and the development of asymmetric acid efflux in gravistimulated roots.

  7. Anaerobic microbial community response to methanogenic inhibitors 2-bromoethanesulfonate and propynoic acid.

    PubMed

    Webster, Tara M; Smith, Adam L; Reddy, Raghav R; Pinto, Ameet J; Hayes, Kim F; Raskin, Lutgarde

    2016-08-01

    Methanogenic inhibitors are often used to study methanogenesis in complex microbial communities or inhibit methanogens in the gastrointestinal tract of livestock. However, the resulting structural and functional changes in archaeal and bacterial communities are poorly understood. We characterized microbial community structure and activity in mesocosms seeded with cow dung and municipal wastewater treatment plant anaerobic digester sludge after exposure to two methanogenic inhibitors, 2-bromoethanesulfonate (BES) and propynoic acid (PA). Methane production was reduced by 89% (0.5 mmol/L BES), 100% (10 mmol/LBES), 24% (0.1 mmol/LPA), and 95% (10 mmol/LPA). Using modified primers targeting the methyl-coenzyme M reductase (mcrA) gene, changes in mcrA gene expression were found to correspond with changes in methane production and the relative activity of methanogens. Methanogenic activity was determined by the relative abundance of methanogen 16S rRNA cDNA as a percentage of the total community 16S rRNA cDNA. Overall, methanogenic activity was lower when mesocosms were exposed to higher concentrations of both inhibitors, and aceticlastic methanogens were inhibited to a greater extent than hydrogenotrophic methanogens. Syntrophic bacterial activity, measured by 16S rRNA cDNA, was also reduced following exposure to both inhibitors, but the overall structure of the active bacterial community was not significantly affected. PMID:26987552

  8. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  9. Removal and upgrading of lignocellulosic fermentation inhibitors by in situ biocatalysis and liquid-liquid extraction.

    PubMed

    Tomek, Kyle J; Saldarriaga, Carlos Rafael Castillo; Velasquez, Fernando Peregrino Cordoba; Liu, Tongjun; Hodge, David B; Whitehead, Timothy A

    2015-03-01

    Hydroxycinnamic acids are known to inhibit microbial growth during fermentation of lignocellulosic biomass hydrolysates, and the ability to diminish hydroxycinnamic acid toxicity would allow for more effective biological conversion of biomass to fuels and other value-added products. In this work, we provide a proof-of-concept of an in situ approach to remove these fermentation inhibitors through constituent expression of a phenolic acid decarboxylase combined with liquid-liquid extraction of the vinyl phenol products. As a first step, we confirmed using simulated fermentation conditions in two model organisms, Escherichia coli and Saccharomyces cerevisiae, that the product 4-vinyl guaiacol is more inhibitory to growth than ferulic acid. Partition coefficients of ferulic acid, p-coumaric acid, 4-vinyl guaiacol, and 4-ethyl phenol were measured for long-chain primary alcohols and alkanes, and tetradecane was identified as a co-solvent that can preferentially extract vinyl phenols relative to the acid parent and additionally had no effect on microbial growth rates or ethanol yields. Finally, E. coli expressing an active phenolic acid decarboxylase retained near maximum anaerobic growth rates in the presence of ferulic acid if and only if tetradecane was added to the fermentation broth. This work confirms the feasibility of donating catabolic pathways into fermentative microorganisms in order to ameliorate the effects of hydroxycinnamic acids on growth rates, and suggests a general strategy of detoxification by simultaneous biological conversion and extraction. PMID:25311910

  10. Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

    PubMed

    Bernard, Dannie; Gebbia, Marinella; Prabha, Swayam; Gronda, Marcela; MacLean, Neil; Wang, Xiaoming; Hurren, Rose; Sukhai, Mahadeo A; Cho, Eunice E; Manolson, Morris F; Datti, Alessandro; Wrana, Jeffrey; Minden, Mark D; Al-Awar, Rima; Aman, Ahmed; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D

    2015-07-01

    To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function. PMID:25832785

  11. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki

    2014-10-13

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

  12. Novel quinolinonyl diketo acid derivatives as HIV-1 integrase inhibitors: design, synthesis, and biological activities.

    PubMed

    Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Miele, Gaetano; Crucitti, Giuliana Cuzzucoli; Iacovo, Alberto; Rosi, Federica; Lavecchia, Antonio; Marinelli, Luciana; Di Giovanni, Carmen; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

    2008-08-14

    Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3'-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN-DNA acceptor site without affecting the DNA donor site.

  13. Search for novel histone deacetylase inhibitors. Part II: design and synthesis of novel isoferulic acid derivatives.

    PubMed

    Lu, Wen; Wang, Fang; Zhang, Tao; Dong, Jinyun; Gao, Hongping; Su, Ping; Shi, Yaling; Zhang, Jie

    2014-05-01

    Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDACIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited significant enzymatic inhibitory activities, with IC50 values of 0.73 ± 0.08 and 0.57 ± 0.16 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells. Especially, IF6 displayed promising profile as an antitumor candidate with IC50 value of 3.91 ± 0.97 μM against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising lead compounds for further optimization. PMID:24702857

  14. Development of novel ferulic acid derivatives as potent histone deacetylase inhibitors.

    PubMed

    Wang, Fang; Lu, Wen; Zhang, Tao; Dong, Jinyun; Gao, Hongping; Li, Pengfei; Wang, Sicen; Zhang, Jie

    2013-11-15

    Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety (SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6 and FA16 exhibited significant enzymatic inhibitory activities, with IC50 values of 3.94 and 2.82 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated that these ferulic acid derivatives could serve as promising lead compounds for further optimization. PMID:24095016

  15. [Prevention of colorectal cancer: acetylsalicyclic acid and cyclo-oxygenase-2 inhibitors are only partially effective].

    PubMed

    Kleibeuker, J H; de Vries, E G

    2003-11-01

    Acetylsalicylic acid was recently shown to inhibit the development of colorectal adenomas in subjects with a moderately increased risk for colorectal cancer. The mechanisms by which acetylsalicylic acid and other NSAIDs, including COX-2 inhibitors, exert this effect include: inhibition of COX-2, induction of apoptosis and induction of the P21 protein that controls the development of crypt cells. For the majority of the population, a healthy lifestyle and healthy eating habits are the best means of preventing colorectal cancer. In addition, population-wide screening should be encouraged. For people with an increased risk of colorectal cancer, acetylsalicylic acid only has a partial effect and therefore endoscopic surveillance is still indicated. Sulindac or celecoxib may be useful for patients with familial adenomatous polyposis and in particular for inhibiting the development of rectal adenomas after subtotal colectomy with ileorectal anastomosis. However, in these cases endoscopic surveillance is also still necessary.

  16. Discovery of wall teichoic acid inhibitors as potential anti-MRSA β-lactam combination agents.

    PubMed

    Wang, Hao; Gill, Charles J; Lee, Sang H; Mann, Paul; Zuck, Paul; Meredith, Timothy C; Murgolo, Nicholas; She, Xinwei; Kales, Susan; Liang, Lianzhu; Liu, Jenny; Wu, Jin; Santa Maria, John; Su, Jing; Pan, Jianping; Hailey, Judy; Mcguinness, Debra; Tan, Christopher M; Flattery, Amy; Walker, Suzanne; Black, Todd; Roemer, Terry

    2013-02-21

    Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

  17. Docking analysis of gallic acid derivatives as HIV-1 protease inhibitors.

    PubMed

    Singh, Anjali; Pal, Tapan Kumar

    2015-01-01

    HIV-1 Protease (HIV-1 PR) enzymes are essential for accurate assembly and maturation of infectious HIV retroviruses. The significant role of HIV-1 protease in viral replication has made it a potential drug target. In the recent past, phytochemical Gallic Acid (GA) derivatives have been screened for protease inhibitor activity. The present work aims to design and evaluate potential GA-based HIV-1 PR phytoinhibitors by docking approach. The ligands were prepared by ChemDraw and docking was performed in HEX software. In this present study, one of the GA analogues (GA4) emerged as a potent drug candidate for HIV-1 PR inhibition, and docking results showed it to be comparable with anti-HIV drugs, darunavir and amprenavir. The GA4 derivative provided a lead for designing more effective HIV-1 PR inhibitors.

  18. Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana.

    PubMed

    Jiang, He Zhong; Quan, Xiao Fang; Tian, Wei Xi; Hu, Jiang Miao; Wang, Peng Cheng; Huang, Sheng Zhuo; Cheng, Zhong Quan; Liang, Wen Juan; Zhou, Jun; Ma, Xiao Feng; Zhao, You Xing

    2010-10-15

    Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1-13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC(50) values ranging from 1.24 to 91.07 μM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors.

  19. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    SciTech Connect

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  20. The Ornithine Decarboxylase Gene of Caenorhabditis Elegans: Cloning, Mapping and Mutagenesis

    PubMed Central

    Macrae, M.; Plasterk, RHA.; Coffino, P.

    1995-01-01

    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 422 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5' RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved stage 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. PMID:7498733

  1. The ornithine decarboxylase gene of Caenorhabditis elegans: Cloning, mapping and mutagenesis

    SciTech Connect

    Macrae, M.; Coffino, P.; Plasterk, R.H.A.

    1995-06-01

    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 442 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5{prime} RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. 37 refs., 6 figs., 1 tab.

  2. Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor.

    PubMed

    Kalyanavenkataraman, Subhalakshmi; Nanjan, Pandurangan; Banerji, Asoke; Nair, Bipin G; Kumar, Geetha B

    2016-06-01

    Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9μM to as high as 333μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA. PMID:27038848

  3. A defect in pyruvate decarboxylase in a child with an intermittent movement disorder

    PubMed Central

    Blass, John P.; Avigan, Joel; Uhlendorf, B. William

    1970-01-01

    A patient with an intermittent movement disorder has been found to have an inherited defect in pyruvate decarboxylase ((2-oxo-acid carboxy-lyase, E.C. 4.1.1.1.). The patient is a 9 yr old boy who since infancy has had repeated episodes of a combined cerebellar and choreoathetoid movement disorder. He has an elevated level of pyruvic acid in his blood, an elevated urinary alanine content, and less marked elevations in blood alanine and lactate. Methods were developed to study his metabolic abnormality in dilute suspensions of white blood cells and cultured skin fibroblasts, as well as in cell-free sonicates of fibroblasts. Oxidation of pyruvic acid-1-14C and pyruvic acid-2-14C by his cells and pyruvate decarboxylase activity in sonicates of his cells were less than 20% of those in cells from control subjects. Oxidation of glutamic acid-U-14C, acetate-1-14C, and palmitate-1-14C was normal, as was incorporation of alanine-U-14C into protein. The rate of oxidation of pyruvic acid by the father's cells and the activity of pyruvate decarboxylase in the father's sonicated fibroblasts were intermediate between those of the patient and those of controls. Values for the mother were at or just below the lower limits of the ranges in controls. Kinetic data suggested the posibility of several forms of pyruvate decarboxylase in this family. Possible mechanisms relating the chemical abnormality and the clinical symptoms in this patient are discussed. PMID:4313434

  4. The intracellular Ca(2+)-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes.

    PubMed

    Cheng, T C; Benton, H P

    1994-07-15

    Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca(2+)-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [35S]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [35S]methionine-labelled gels. The alterations in protein synthesis induced by Ca(2+)-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca(2+)-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca(2+)-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca(2+)-pump inhibitors such as TG. PMID:8043004

  5. Angiotensin-converting enzyme inhibitors modulate kynurenic acid production in rat brain cortex in vitro.

    PubMed

    Zakrocka, Izabela; Turski, Waldemar A; Kocki, Tomasz

    2016-10-15

    It is well established that the renin-angiotensin system (RAS) is present in the brain and that glutamate activates the brain centers responsible for blood pressure control. An antagonist of glutamate, kynurenic acid (KYNA) was shown to decrease blood pressure after intracerebral administration. KYNA is an endogenous metabolite of tryptophan produced from the breakdown of kynurenine by kynurenine aminotransferases (KAT), mainly within astrocytes. The purpose of this study was to evaluate the influence of three angiotensin-converting enzyme inhibitors (lisinopril, perindopril and ramipril) on KYNA production and KAT activity in the rat brain cortex in vitro. The effect of the angiotensin-converting enzyme inhibitors on KYNA production was examined on rat brain cortical slices incubated for 2h in the presence of l-kynurenine and the angiotensin-converting enzyme inhibitors. To analyze KAT I and KAT II activity, brain cortical homogenates were incubated for 2h with L-kynurenine and the tested drugs. KYNA was separated by HPLC and quantified fluorometrically. Among the examined angiotensin-converting enzyme inhibitors, lisinopril increased KYNA production, perindopril was ineffective, and ramipril decreased KYNA synthesis in rat brain cortical slices. Lisinopril increased KAT I activity and perindopril did not affect it. However, ramipril lowered KAT I activity in rat brain cortex in vitro. Neither lisinopril nor perindopril affected KAT II activity, but ramipril decreased KAT II activity in the rat brain cortex in vitro. Our study reveals that angiotensin-converting enzyme inhibitors show various influences on KYNA production in rat brain cortical slices and activity of KATs.

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

    PubMed

    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.

  7. Glycine decarboxylase controls photosynthesis and plant growth.

    PubMed

    Timm, Stefan; Florian, Alexandra; Arrivault, Stephanie; Stitt, Mark; Fernie, Alisdair R; Bauwe, Hermann

    2012-10-19

    Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO(2) acceptor ribulose 1,5-bisphosphate indicated higher drain from CO(2) fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.

  8. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    PubMed

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  9. Synthesis and characterization of phosphocitric acid, a potent inhibitor of hydroxylapatite crystal growth.

    PubMed

    Tew, W P; Mahle, C; Benavides, J; Howard, J E; Lehninger, A L

    1980-04-29

    Human urine and extracts of rat liver mitochondria contain apparently identical agents capable of inhibiting the precipitation or crystallization of calcium phosphate. Its general properties, as well as 1H NMR and mass spectra, have suggested that the agent is phosphocitric acid. This paper reports the synthesis of phosphocitric acid via the phosphorylation of triethyl citrate with o-phenylene phosphochloridate, hydrogenolysis of the product to yield triethyl phosphocitrate, hydrolytic removal of the blocking ethyl groups and also chromatographic purification. An enzymatic assay of phosphocitrate is described. Synthetic phosphocitrate was found to be an exceedingly potent inhibitor of the growth of hydroxylapatite seed crystals in a medium supersaturated with respect to Ca2+ and phosphate. Comparative assays showed phosphocitrate to be much more potent than the most active precipitation-crystallization inhibitors previously reported, which include pyrophosphate and ATP. 14C-Labeled phosphocitrate was bound very tightly to hydroxylapatite crystals. Such binding appeared to be essential for its inhibitory activity on crystal growth. Citrate added before but not after, phosphocitrate greatly enhanced the inhibitory potency of the latter. This enhancement effect was not given by other tricarboxylic acids. The monoethyl ester of phosphocitrate had no inhibitory effect on hydroxylapatite crystal growth.

  10. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

    PubMed

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual

    2016-01-01

    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

  11. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors

    PubMed Central

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual

    2016-01-01

    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C. PMID:27391465

  12. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

    PubMed

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual

    2016-01-01

    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C. PMID:27391465

  13. Activity of cefazolin and two beta-lactamase inhibitors, clavulanic acid and sulbactam, against Bacteroides fragilis.

    PubMed Central

    Fekete, T; McGowen, J; Cundy, K R

    1987-01-01

    One hundred clinical isolates of the Bacteroides fragilis group of bacteria were tested by agar dilution for susceptibility to cefazolin alone or in combination with clavulanic acid or sulbactam. For cefazolin, the MIC for 50% of the isolates (MIC50) was 32 micrograms/ml, the breakpoint for susceptibility. With the addition of 0.5 micrograms of clavulanic acid per ml, the MIC for 90% of the isolates (MIC90) was 8 micrograms/ml, well within the achievable range of concentrations in serum or tissue. Similarly, with the addition of 0.5 micrograms of sulbactam per ml, the MIC90 was 16 micrograms/ml. The addition of a higher concentration (4.0 micrograms/ml) of clavulanic acid or sulbactam resulted in MIC90S which were fourfold lower than those with 0.5 micrograms/ml. A fixed ratio of cefazolin-beta-lactamase inhibitor of 4:1 resulted in an MIC50 and MIC90 which were intermediate between the 0.5- and 4.0-micrograms/ml fixed concentration of beta-lactamase inhibitor. PMID:3032097

  14. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    SciTech Connect

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  15. Benzimidazole as corrosion inhibitor for heat treated 6061 Al- SiCp composite in acetic acid

    NASA Astrophysics Data System (ADS)

    Chacko, Melby; Nayak, Jagannath

    2015-06-01

    6061 Al-SiCpcomposite was solutionizedat 350 °C for 30 minutes and water quenched. It was then underaged at 140 °C (T6 treatment). The aging behaviour of the composite was studied using Rockwell B hardness measurement. Corrosion behaviour of the underaged sample was studied in different concentrations of acetic acid and at different temperatures. Benzimidazole at different concentrations was used for the inhibition studies. Inhibition efficiency of benzimidazole was calculated for different experimental conditions. Thermodynamic parameters were found out which suggested benzimidazole is an efficient inhibitor and it adsorbed on to the surface of composite by mixed adsorption where chemisorption is predominant.

  16. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    PubMed

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P

    1987-02-15

    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

  17. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    PubMed Central

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P

    1987-01-01

    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed. PMID:3297044

  18. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  19. Epigenetic suppression of the antitumor cytotoxicity of NK cells by histone deacetylase inhibitor valproic acid

    PubMed Central

    Shi, Xiumin; Li, Min; Cui, Meizi; Niu, Chao; Xu, Jianting; Zhou, Lei; Li, Wei; Gao, Yushun; Kong, Weisheng; Cui, Jiuwei; Hu, Jifan; Jin, Haofan

    2016-01-01

    Natural killer (NK) cells play an essential role in the fight against tumor development. The therapeutic use of autologous NK cells has been exploited to treat human malignancies, yet only limited antitumor activity is observed in cancer patients. In this study, we sought to augment the antitumor activity of NK cells using epigenetic approaches. Four small molecules that have been known to promote epigenetic reprogramming were tested for their ability to enhance the activity of NK cells. Using a tumor cell lysis assay, we found that the DNA demethylating agent 5-azacytidine and vitamin C did not significantly affect the tumor killing ability of NK cells. The thyroid hormone triiodothyronine (T3) slightly increased the activity of NK cells. The histone deacetylase inhibitor valproic acid (VPA), however, inhibited NK cell lytic activity against leukemic cells in a dose-dependent manner. Pretreatment using VPA reduced IFNγ secretion, impaired CD107a degranulation, and induced apoptosis by activating the PD-1/PD-L1 pathway. VPA downregulated the expression of the activating receptor NKG2D (natural-killer group 2, member D) by inducing histone K9 hypermethylation and DNA methylation in the gene promoter. Histone deacetylase inhibitors have been developed as anticancer agents for use as monotherapies or in combination with other anticancer therapies. Our data suggest that the activity of histone deacetylase inhibitors on NK cell activity should be considered in drug development. PMID:27152238

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

    PubMed

    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.

  1. Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

    SciTech Connect

    Lichtenthaler, H.K.; Kobek, K. )

    1989-04-01

    Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from {sup 14}C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis.

  2. Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-05-03

    The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product ..gamma..-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 /sup 0/C, the isotope effect is k/sup 14//k/sup 15/ = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is K/sup 14//K/sup 15/ - 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.

  3. Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1).

    PubMed

    Orchard, Michael G; Neuss, Judi C; Galley, Carl M S; Carr, Andrew; Porter, David W; Smith, Phillip; Scopes, David I C; Haydon, David; Vousden, Katherine; Stubberfield, Colin R; Young, Kate; Page, Martin

    2004-08-01

    The first inhibitors of fungal protein: mannosyl transferase 1 (PMT1) are described. They are based upon rhodanine-3-acetic acid and several compounds have been identified, for example, 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (5a), which inhibit Candida albicans PMT1 with IC(50)s in the range 0.2-0.5 microM. Members of the series are effective in inducing changes in morphology of C. albicans in vitro that have previously been associated with loss of the transferase activity. These compounds could serve as useful tools for studying the effects of protein O-mannosylation and its relevance in the search for novel antifungal agents. PMID:15225710

  4. Hydrophobic amino acids as a new class of kinetic inhibitors for gas hydrate formation

    PubMed Central

    Sa, Jeong-Hoon; Kwak, Gye-Hoon; Lee, Bo Ram; Park, Da-Hye; Han, Kunwoo; Lee, Kun-Hong

    2013-01-01

    As the foundation of energy industry moves towards gas, flow assurance technology preventing pipelines from hydrate blockages becomes increasingly significant. However, the principle of hydrate inhibition is still poorly understood. Here, we examined natural hydrophobic amino acids as novel kinetic hydrate inhibitors (KHIs), and investigated hydrate inhibition phenomena by using them as a model system. Amino acids with lower hydrophobicity were found to be better KHIs to delay nucleation and retard growth, working by disrupting the water hydrogen bond network, while those with higher hydrophobicity strengthened the local water structure. It was found that perturbation of the water structure around KHIs plays a critical role in hydrate inhibition. This suggestion of a new class of KHIs will aid development of KHIs with enhanced biodegradability, and the present findings will accelerate the improved control of hydrate formation for natural gas exploitation and the utilization of hydrates as next-generation gas capture media. PMID:23938301

  5. Hydrophobic amino acids as a new class of kinetic inhibitors for gas hydrate formation

    NASA Astrophysics Data System (ADS)

    Sa, Jeong-Hoon; Kwak, Gye-Hoon; Lee, Bo Ram; Park, Da-Hye; Han, Kunwoo; Lee, Kun-Hong

    2013-08-01

    As the foundation of energy industry moves towards gas, flow assurance technology preventing pipelines from hydrate blockages becomes increasingly significant. However, the principle of hydrate inhibition is still poorly understood. Here, we examined natural hydrophobic amino acids as novel kinetic hydrate inhibitors (KHIs), and investigated hydrate inhibition phenomena by using them as a model system. Amino acids with lower hydrophobicity were found to be better KHIs to delay nucleation and retard growth, working by disrupting the water hydrogen bond network, while those with higher hydrophobicity strengthened the local water structure. It was found that perturbation of the water structure around KHIs plays a critical role in hydrate inhibition. This suggestion of a new class of KHIs will aid development of KHIs with enhanced biodegradability, and the present findings will accelerate the improved control of hydrate formation for natural gas exploitation and the utilization of hydrates as next-generation gas capture media.

  6. Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

    PubMed

    Shui, Zong-Xia; Qin, Han; Wu, Bo; Ruan, Zhi-yong; Wang, Lu-shang; Tan, Fu-Rong; Wang, Jing-Li; Tang, Xiao-Yu; Dai, Li-Chun; Hu, Guo-Quan; He, Ming-Xiong

    2015-07-01

    Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

  7. Identification of KMU-3, a novel derivative of gallic acid, as an inhibitor of adipogenesis.

    PubMed

    Park, Yu-Kyoung; Lee, Jinho; Hong, Victor Sukbong; Choi, Jong-Soon; Lee, Tae-Yoon; Jang, Byeong-Churl

    2014-01-01

    Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. Excessive adipogenesis, however, is largely linked to the development of obesity. Herein we investigated a library of 53 novel chemicals, generated from a number of polyphenolic natural compounds, on adipogenesis. Strikingly, among the chemicals tested, KMU-3, a derivative of gallic acid, strongly suppressed lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, KMU-3 inhibited expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) during adipocyte differentiation. Moreover, KMU-3 reduced expressions of adipokines, including retinol binding protein-4 (RBP-4), leptin, and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. Of further note, KMU-3 rapidly blocked the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) during the early stage of adipogenesis. Importantly, pharmacological inhibition studies revealed that AG490, a JAK-2/STAT-3 inhibitor suppressed adipogenesis and STAT-3 phosphorylation, implying that early blockage of STAT-3 activity is crucial for the KMU-3-mediated anti-adipogenesis. These findings demonstrate firstly that KMU-3 inhibits adipogenesis by down-regulating STAT-3, PPAR-γ, C/EBP-α, and FAS. This work shows that KMU-3 is an inhibitor of adipogenesis and thus may have therapeutic potential against obesity.

  8. The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors

    PubMed Central

    Binder, Veronika; Ljubojevic, Senka; Haybaeck, Johannes; Holzer, Michael; El-Gamal, Dalia; Schicho, Rudolf; Pieske, Burkert; Heinemann, Akos; Marsche, Gunther

    2014-01-01

    Objective Elevated levels of advanced oxidation protein products (AOPPs) have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis and atherosclerosis. Recent findings revealed that AOPPs are inhibitors of the major high-density lipoprotein (HDL) receptor, scavenger receptor class B, type 1 (SR-BI). Here we investigated what oxidation induced structural alterations convert plasma albumin into an HDL-receptor inhibitor. Approach and Results Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high affinity SR-BI ligands. Protection of albumin lysine-residues prior exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin lysine-residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of HDL. Conclusion Given that several potential atheroprotective activities of HDL are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease. PMID:23493288

  9. Click Chemistry in Lead Optimization of Boronic Acids as β-Lactamase Inhibitors.

    PubMed

    Caselli, Emilia; Romagnoli, Chiara; Vahabi, Roza; Taracila, Magdalena A; Bonomo, Robert A; Prati, Fabio

    2015-07-23

    Boronic acid transition-state inhibitors (BATSIs) represent one of the most promising classes of β-lactamase inhibitors. Here we describe a new class of BATSIs, namely, 1-amido-2-triazolylethaneboronic acids, which were synthesized by combining the asymmetric homologation of boronates with copper-catalyzed azide-alkyne cycloaddition for the stereoselective insertion of the amido group and the regioselective formation of the 1,4-disubstituted triazole, respectively. This synthetic pathway, which avoids intermediate purifications, proved to be flexible and efficient, affording in good yields a panel of 14 BATSIs bearing three different R1 amide side chains (acetamido, benzylamido, and 2-thienylacetamido) and several R substituents on the triazole. This small library was tested against two clinically relevant class C β-lactamases from Enterobacter spp. and Pseudomonas aeruginosa. The K(i) value of the best compound (13a) was as low as 4 nM with significant reduction of bacterial resistance to the combination of cefotaxime/13a. PMID:26102369

  10. Pharmacodynamic effects of antibiotics and acid pump inhibitors on Helicobacter pylori.

    PubMed Central

    Sörberg, M; Hanberger, H; Nilsson, M; Nilsson, L E

    1997-01-01

    Pharmacodynamic studies of Helicobacter pylori exposed to amoxicillin, clarithromycin, metronidazole, omeprazole, and lansoprazole were performed with microscopy, viable count determination, and bioluminescence assay of intracellular ATP. The pharmacodynamic parameters determined were change in morphology, change in cell density, postantibiotic effect (PAE), and control-related effective regrowth time (CERT). The PAE is delayed regrowth after brief exposure to antibiotics or acid pump inhibitors. CERT was defined as the time required for the bacteria to resume logarithmic growth and return to the pre-exposure inoculum in the test culture minus the corresponding time for the control culture. CERT measures the combined effect of initial killing and PAE. There was a good concordance between the bioluminescence assay and viable counts for determining CERT, which makes this parameter useful for pharmacodynamic studies of the effects of antibiotics and acid pump inhibitors on H. pylori. Amoxicillin and metronidazole produced a strong, concentration-dependent initial decrease in CFU per milliliter, but there was a less prominent initial change in intracellular ATP in these cultures. Amoxicillin caused a long PAE when assayed by the bioluminescence assay but no PAE or a negative PAE when assayed by viable count determination. However, amoxicillin showed similar long CERTs with both methods. The pharmacodynamic effects of amoxicillin were concentration dependent up to a maximum response, indicating that concentrations above this level do not increase the antibiotic effect. The PAEs and CERTs of clarithromycin and metronidazole were concentration dependent with no maximum response. With omeprazole and lanzoprazole, there was no PAE or CERT. PMID:9333051

  11. Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-skin Cancer Effects of Ursolic Acid

    PubMed Central

    Junco, Jacob J.; Mancha, Anna; Malik, Gunjan; Wei, Sung-Jen; Kim, Dae Joon; Liang, Huiyun; Slaga, Thomas J.

    2013-01-01

    Ursolic acid (UA), present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to UA treatment. Herein, skin carcinoma cells (Ca3/7) as compared to skin papilloma cells (MT1/2) displayed more resistance to UA-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in pre-clinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate Rhodamine 123 (Rh123) indicating UA is transported by P-gp. To determine if P-gp inhibition can enhance UA-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil (VRP) or cyclosporin A (CsA). Alternatively, cells were pre-treated with the natural compound resveratrol (RES), a known chemotherapy sensitizer. VRP and RES enhanced the effects of UA in both cell lines, while CsA only did so in Ca3/7 cells. Similarly, VRP inhibited Rh123 efflux in both lines, while CsA only inhibited Rh123 efflux in Ca3/7 cells. RES did not inhibit Rh123 efflux in either line, indicating the synergistic effects of RES and UA are not manifest by inhibition of P-gp-mediated efflux of UA. These results indicate that the anti-skin cancer effects of UA are enhanced with P-gp inhibitors. In addition, RES and UA interact synergistically, but not through inhibition of P-gp. Implications Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen. PMID:24072817

  12. Polyamine formation by arginine decarboxylase as a transducer of hormonal, environmental and stress stimuli in higher plants

    NASA Technical Reports Server (NTRS)

    Galston, A. W.; Flores, H. E.; Kaur-Sawhney, R.

    1982-01-01

    Recent evidence implicates polyamines including putrescine in the regulation of such diverse plant processes as cell division, embryogenesis and senescence. We find that the enzyme arginine decarboxylase, which controls the rate of putrescine formation in some plant systems, is activated by light acting through P(r) phytochrome as a receptor, by the plant hormone gibberellic acid, by osmotic shock and by other stress stimuli. We therefore propose arginine decarboxylase as a possible transducer of the various initially received tropistic stimuli in plants. The putrescine formed could act by affecting cytoskeletal components.

  13. Dianils: New and effective corrosion inhibitors for oil-well steel (N-80) and mild steel in boiling hydrochloric acid

    SciTech Connect

    Quraishi, M.A.; Jamal, D.

    2000-02-01

    Selected condensation products of aromatic aldehydes and p-phenylenediamine have been synthesized and evaluated as corrosion inhibitors for mild steel (MS) and oil-well steel (N-80) in 15% hydrochloric acid (HCl) at 105 C {+-} 2 C by the weight loss method. All the condensation products showed excellent performance. 2,4-dicinnamyledene aminophenylene (DCAP) was found to be the best corrosion inhibitor. It exhibited 99.75% inhibition efficiency (IE) for MS and 99.12% for N-80 steel at 5,000 ppm of inhibitor concentration. The potentiodynamic polarization studies carried out at room temperature on MS in 15% HCl containing 500 ppm of condensation products showed that all the investigated compounds were mixed type inhibitors, whereas 500 ppm DCAP on N-80 steel behaved predominantly as anodic inhibitors. The adsorption of all the condensation products was found to obey Temkin's adsorption isotherm.

  14. Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli.

    PubMed Central

    Canellakis, E S; Paterakis, A A; Huang, S C; Panagiotidis, C A; Kyriakidis, D A

    1993-01-01

    The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E. coli genomic library. A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced. The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme. Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator. The open reading frame next to antizyme is homologous to sensor kinases. Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels. Extracts from E. coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity. These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes. Images Fig. 2 Fig. 4 Fig. 6 PMID:8346225

  15. Absence of malonyl coenzyme A decarboxylase in mice increases cardiac glucose oxidation and protects the heart from ischemic injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acute pharmacological inhibition of cardiac malonyl coenzyme A decarboxylase (MCD) protects the heart from ischemic damage by inhibiting fatty acid oxidation and stimulating glucose oxidation. However, it is unknown whether chronic inhibition of MCD results in altered cardiac function, energy metabo...

  16. Theoretical study of inhibition efficiencies of some amino acids on corrosion of carbon steel in acidic media: green corrosion inhibitors.

    PubMed

    Dehdab, Maryam; Shahraki, Mehdi; Habibi-Khorassani, Sayyed Mostafa

    2016-01-01

    Inhibition efficiencies of three amino acids [tryptophan (B), tyrosine (c), and serine (A)] have been studied as green corrosion inhibitors on corrosion of carbon steel using density functional theory (DFT) method in gas and aqueous phases. Quantum chemical parameters such as EH OMO (highest occupied molecular orbital energy), E LUMO (lowest unoccupied molecular orbital energy), hardness (η), polarizability ([Formula: see text]), total negative charges on atoms (TNC), molecular volume (MV) and total energy (TE) have been calculated at the B3LYP level of theory with 6-311++G** basis set. Consistent with experimental data, theoretical results showed that the order of inhibition efficiency is tryptophan (B) > tyrosine (C) > serine (A). In order to determine the possible sites of nucleophilic and electrophilic attacks, local reactivity has been evaluated through Fukui indices.

  17. Discovery of FDA-approved drugs as inhibitors of fatty acid binding protein 4 using molecular docking screening.

    PubMed

    Wang, Yan; Law, Wai-Kit; Hu, Jian-Shu; Lin, Huang-Quan; Ip, Tsz-Ming; Wan, David Chi-Cheong

    2014-11-24

    We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking screening from FDA-approved drugs. Subsequently, the biochemical characterizations showed that levofloxacin directly inhibited FABP4 activity in both the in vitro ligand displacement assay and cell-based function assay. Furthermore, levofloxacin did not induce adipogenesis in adipocytes, which is the major adverse effect of FABP4 inhibitors.

  18. Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase▿

    PubMed Central

    Sharma, Shilpi; Sharma, Shailendra Kumar; Modak, Rahul; Karmodiya, Krishanpal; Surolia, Namita; Surolia, Avadhesha

    2007-01-01

    The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (−)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite. PMID:17485508

  19. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    SciTech Connect

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P.

    2010-06-14

    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  20. Functional analysis of cis-aconitate decarboxylase and trans-aconitate metabolism in riboflavin-producing filamentous Ashbya gossypii.

    PubMed

    Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2014-05-01

    In Ashbya gossypii, isocitrate lyase (ICL1) is a very crucial enzyme for riboflavin production. Itaconate, the inhibitor of ICL1, has been used as an antimetabolite for mutagenic studies in A. gossypii. It has been reported that itaconate is produced from cis-aconitate by cis-aconitate decarboxylase (CAD1) in Aspergillus terreus. In this study, identification of CAD1 gene and determination of the presence of itaconate in the riboflavin biosynthetic pathway in A. gossypii were carried out to confirm itaconate metabolism. Although no CAD1 candidate gene was found and no itaconate production was observed, cis- and trans-aconitate were detected in the riboflavin production phase. It is known that trans-aconitate inhibits aconitase (ACO1) in the tricarboxylic acid cycle. In A. gossypii, the transcription level of AGR110Wp, the homolog of trans-aconitate 3-methyltransferase (TMT1), was enhanced by almost threefold during riboflavin production than that during its growth phase. TMT1 catalyzes the methylation reaction of trans-aconitate in Saccharomyces cerevisiae. Thus, these results suggest that the enhancement of the transcription level of this TMT1 homolog decreases the trans-aconitate level, which may mitigate the inhibition of ACO1 by oxidative stress in the riboflavin biosynthetic pathway in A. gossypii. This is a novel finding in A. gossypii, which may open new metabolic engineering ideas for improving riboflavin productivity. PMID:24315530

  1. Concentrated sulfite-yeast fermenting mixture as a corrosion inhibitor of copper in mixtures of sulfuric and nitric acid

    SciTech Connect

    Agaev, N.M.; Smorodin, A.E.; Rzaev, E.R.; Tyr, S.G.; Shlimak, Ya.B.; Geidarova, G.D.; Eremeeva, R.A.; Nasirov, G.N.

    1987-03-01

    At the Baku factory of residential air conditioning systems both preliminary and final pickling of copper tubing is carried out in a solution of sulfuric and nitric acids. The authors of this study, in seeking an inhibitor to control this process, evaluate the protective properties of an inhibitor based on a concentrated sulfite-yeast fermenting mixture that is generated as a common waste product by the cellulose-pulp industry. It consists of calcium, sodium, and ammonium salts of lignin sulfonic acids. Tests revealed not only its inhibiting effectiveness but also its capacity to lower toxic gas levels of nitrogen oxides in the plant environment.

  2. Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms.

    PubMed

    Goodman, S A; Esau, B; Koontz, J W

    1988-11-01

    Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

  3. Structure activity relationship of phenolic acid inhibitors of α-synuclein fibril formation and toxicity

    PubMed Central

    Ardah, Mustafa T.; Paleologou, Katerina E.; Lv, Guohua; Abul Khair, Salema B.; Kazim, Abdulla S.; Minhas, Saeed T.; Al-Tel, Taleb H.; Al-Hayani, Abdulmonem A.; Haque, Mohammed E.; Eliezer, David; El-Agnaf, Omar M. A.

    2014-01-01

    The aggregation of α-synuclein (α-syn) is considered the key pathogenic event in many neurological disorders such as Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, giving rise to a whole category of neurodegenerative diseases known as synucleinopathies. Although the molecular basis of α-syn toxicity has not been precisely elucidated, a great deal of effort has been put into identifying compounds that could inhibit or even reverse the aggregation process. Previous reports indicated that many phenolic compounds are potent inhibitors of α-syn aggregation. The aim of the present study was to assess the anti-aggregating effect of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a benzoic acid derivative that belongs to a group of phenolic compounds known as phenolic acids. By employing an array of biophysical and biochemical techniques and a cell-viability assay, GA was shown not only to inhibit α-syn fibrillation and toxicity but also to disaggregate preformed α-syn amyloid fibrils. Interestingly, GA was found to bind to soluble, non-toxic oligomers with no β-sheet content, and to stabilize their structure. The binding of GA to the oligomers may represent a potential mechanism of action. Additionally, by using structure activity relationship data obtained from fourteen structurally similar benzoic acid derivatives, it was determined that the inhibition of α-syn fibrillation by GA is related to the number of hydroxyl moieties and their position on the phenyl ring. GA may represent the starting point for designing new molecules that could be used for the treatment of PD and related disorders. PMID:25140150

  4. Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors.

    PubMed

    Tangherlini, Giovanni; Torregrossa, Tullio; Agoglitta, Oriana; Köhler, Jens; Melesina, Jelena; Sippl, Wolfgang; Holl, Ralph

    2016-03-01

    Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (Ki=230nM) and (S)-7b (Ki=390nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.

  5. Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

    PubMed

    Groen, B W; van der Meer, R A; Duine, J A

    1988-09-12

    Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

  6. Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4.

    PubMed

    Twahir, Umar T; Stedwell, Corey N; Lee, Cory T; Richards, Nigel G J; Polfer, Nicolas C; Angerhofer, Alexander

    2015-03-01

    This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin-trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion, both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping are similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein. PMID:25526893

  7. Observation of Superoxide Production During Catalysis of Bacillus subtilis Oxalate Decarboxylase at pH4

    PubMed Central

    Twahir, Umar T.; Stedwell, Corey N.; Lee, Cory T.; Richards, Nigel G. J.; Polfer, Nicolas C.; Angerhofer, Alexander

    2015-01-01

    This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping is similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein. PMID:25526893

  8. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

    PubMed

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia

    2014-03-01

    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  9. Study of Ellagic Acid as a Natural Elastase Inhibitor by Spectroscopic Methods

    NASA Astrophysics Data System (ADS)

    Xing, X.; Yang, X.; Cao, Yu.

    2016-03-01

    A new natural inhibitor, ellagic acid (EA), was developed, and its inhibition efficiency on elastase was studied by spectroscopic methods. The experimental results proved that EA is a potent elastase inhibitor with an IC50 value of 1.44 mg/mL by UV-vis spectroscopy, and the inhibition mechanism of elastase was confirmed by fluorescence quenching. The interacting between EA and elastase was mainly based on the static quenching owing to the complex formation when the concentration of EA was ≤40 μM. Fluorescence quenching mainly occurred via dynamic quenching with increasing EA concentration. The thermodynamic parameters such as ΔH and ΔS were calculated to be -86.35 kJ/mol and -165.88 J/mol · K, respectively, indicating that the interactions between EA and elastase were mainly due to van der Waals forces or hydrogen bonding. The synchronous fl uorescence spectra showed that binding of EA to elastase can induce conformational changes in elastase.

  10. Combined kinetic studies and computational analysis on kojic acid analogous as tyrosinase inhibitors.

    PubMed

    Lima, Carlyle Ribeiro; Silva, José Rogério A; de Tássia Carvalho Cardoso, Erica; Silva, Edilene O; Lameira, Jerônimo; do Nascimento, José Luiz Martins; do Socorro Barros Brasil, Davi; Alves, Cláudio N

    2014-01-01

    Tyrosinase is a key enzyme in melanin synthesis and widely distributed in plants and animals tissues. In mammals, this enzyme is related to pigment production, involved in wound healing, primary immune response and it can also contribute to catecholamines synthesis in the brain. Consequently, tyrosinase enzyme represents an attractive and selective target in the field of the medicine, cosmetics and bio-insecticides. In this paper, experimental kinetics and computational analysis were used to study the inhibition of tyrosinase by analogous of Kojic acid. The main interactions occurring between inhibitors-tyrosinase complexes and the influence of divalent cation (Cu2+) in enzymatic inhibition were investigated by using molecular docking, molecular dynamic simulations and electrostatic binding free energy by using the Linear Interaction Energy (LIE) method. The results showed that the electrostatic binding free energy are correlated with values of constant inhibition (r2 = 0.97).Thus, the model obtained here could contribute to future studies of this important system and, therefore, eventually facilitate development of tyrosinase inhibitors. PMID:25004069

  11. Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

    SciTech Connect

    Boggiano, Cesar; Jiang Shibo; Lu Hong; Zhao Qian; Liu Shuwen; Binley, James; Blondelle, Sylvie E. . E-mail: sylvieb@burnham.org

    2006-09-08

    Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide DC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While DC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1{alpha} to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of DC13 implies additional mode(s) of action. These results suggest that DC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.

  12. Arginine Decarboxylase Is Localized in Chloroplasts.

    PubMed Central

    Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

    1995-01-01

    Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

  13. Antibacterial drugs as corrosion inhibitors for bronze surfaces in acidic solutions

    NASA Astrophysics Data System (ADS)

    Rotaru, Ileana; Varvara, Simona; Gaina, Luiza; Muresan, Liana Maria

    2014-12-01

    The present study is aiming to investigate the effect of four antibiotics (amoxicillin, ciprofloxacin, doxycycline and streptomycin,) belonging to different classes of antibacterial drugs on bronze corrosion in a solution simulating an acid rain (pH 4). Due to their ability to form protective films on the metal surface, the tested antibiotics act as corrosion inhibitors for bronze. The antibiotics were tested at various concentrations in order to determine the optimal concentration range for the best corrosion inhibiting effect. In evaluating the inhibition efficiency, polarization curves, electrochemical impedance spectroscopy, SEM and XPS measurements were used. Moreover, a correlation between the inhibition efficiency of some antibacterial drugs and certain molecular parameters was determined by quantum chemical computations. Parameters like energies EHOMO and ELUMO and HOMO-LUMO energy gap were used for correlation with the corrosion data.

  14. Quantitative structure activity relationships of some pyridine derivatives as corrosion inhibitors of steel in acidic medium.

    PubMed

    El Ashry, El Sayed H; El Nemr, Ahmed; Ragab, Safaa

    2012-03-01

    Quantum chemical calculations using the density functional theory (B3LYP/6-31G DFT) and semi-empirical AM1 methods were performed on ten pyridine derivatives used as corrosion inhibitors for mild steel in acidic medium to determine the relationship between molecular structure and their inhibition efficiencies. Quantum chemical parameters such as total negative charge (TNC) on the molecule, energy of highest occupied molecular orbital (E (HOMO)), energy of lowest unoccupied molecular orbital (E (LUMO)) and dipole moment (μ) as well as linear solvation energy terms, molecular volume (Vi) and dipolar-polarization (π) were correlated to corrosion inhibition efficiency of ten pyridine derivatives. A possible correlation between corrosion inhibition efficiencies and structural properties was searched to reduce the number of compounds to be selected for testing from a library of compounds. It was found that theoretical data support the experimental results. The results were used to predict the corrosion inhibition of 24 related pyridine derivatives.

  15. Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase**

    PubMed Central

    Pizzirani, Daniela; Bach, Anders; Realini, Natalia; Armirotti, Andrea; Mengatto, Luisa; Bauer, Inga; Girotto, Stefania; Pagliuca, Chiara; De Vivo, Marco; Summa, Maria; Ribeiro, Alison; Piomelli, Daniele

    2015-01-01

    The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents. PMID:25395373

  16. Rabeprazole: a second-generation proton pump inhibitor in the treatment of acid-related disease.

    PubMed

    Pallotta, Stefano; Pace, Fabio; Marelli, Silvia

    2008-08-01

    Rabeprazole is a proton pump inhibitor (PPI) presenting a very advantageous pharmacodynamic and pharmacokinetic profile over older PPIs. In particular, this drug has a very fast onset of action, due to a short activation time and a very high pKa, and may therefore be defined as a 'second generation' PPI. The aim of this article is to provide an update on the pharmacology and clinical profile of rabeprazole and its use in acid-related disorders, with a particular focus on its role in gastroesophageal reflux disease; in the treatment and prevention of duodenal and gastric ulcers and Zollinger-Ellison syndrome; in the therapy of the extraesophageal manifestations of gastroesophageal reflux disease (in particular the respiratory and ear, nose and throat ones); and in the eradication of Helicobacter pylori.

  17. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

  18. Structural basis for 18-β-glycyrrhetinic acid as a novel non-GSH analog glyoxalase I inhibitor

    PubMed Central

    Zhang, Hong; Huang, Qiang; Zhai, Jing; Zhao, Yi-ning; Zhang, Li-ping; Chen, Yun-yun; Zhang, Ren-wei; Li, Qing; Hu, Xiao-peng

    2015-01-01

    Aim: Glyoxalase I (GLOI), a glutathione (GSH)-dependent enzyme, is overexpressed in tumor cells and related to multi-drug resistance in chemotherapy, making GLOI inhibitors as potential anti-tumor agents. But the most studied GSH analogs exhibit poor pharmacokinetic properties. The aim of this study was to discover novel non-GSH analog GLOI inhibitors and analyze their binding mechanisms. Methods: Mouse GLOI (mGLOI) was expressed in BL21 (DE3) pLysS after induction with isopropyl-β-D-1-thiogalactopyranoside and purified using AKTA FPLC system. An in vitro mGLOI enzyme assay was used to screen a small pool of compounds containing carboxyl groups. Crystal structure of the mGLOI-inhibitor complex was determined at 2.3 Å resolution. Molecular docking study was performed using Discovery Studio 2.5 software package. Results: A natural compound 18-β-glycyrrhetinic acid (GA) and its derivative carbenoxolone were identified as potent competitive non-GSH analog mGLOI inhibitors with Ki values of 0.29 μmol/L and 0.93 μmol/L, respectively. Four pentacyclic triterpenes (ursolic acid, oleanolic acid, betulic acid and tripterine) showed weak activities (mGLOI inhibition ratio <25% at 10 μmol/L) and other three (maslinic acid, corosolic acid and madecassic acid) were inactive. The crystal structure of the mGLOI-GA complex showed that the carboxyl group of GA mimicked the γ-glutamyl residue of GSH by hydrogen bonding to the glutamyl sites (residues Arg38B, Asn104B and Arg123A) in the GSH binding site of mGLOI. The extensive van der Waals interactions between GA and the surrounding residues also contributed greatly to the binding of GA and mGLOI. Conclusion: This work demonstrates a carboxyl group to be an important functional feature of non-GSH analog GLOI inhibitors. PMID:26279158

  19. ANTINOCICEPTIVE EFFECTS OF THE N-ACYLETHANOLAMINE ACID AMIDASE INHIBITOR ARN077 IN RODENT PAIN MODELS

    PubMed Central

    Sasso, Oscar; Moreno-Sanz, Guillermo; Martucci, Cataldo; Realini, Natalia; Dionisi, Mauro; Mengatto, Luisa; Duranti, Andrea; Tarozzo, Glauco; Tarzia, Giorgio; Mor, Marco; Bertorelli, Rosalia; Reggiani, Angelo; Piomelli, Daniele

    2013-01-01

    Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. In the present study, we examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The anti-nociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B-radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α. PMID:23218523

  20. Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea.

    PubMed

    Shon, Yun-Hee; Nam, Kyung-Soo

    2004-01-01

    Polysaccharides (5, 10, 25, 50 and 100 microg ml(-1)) from soybeans and soybeans fermented with Phellinus igniarius or Agrocybe cylindracea inhibited cytochrome P450 1A1, cytochrome P450 1A2 and cytochrome P450 2B1 activities in rat liver microsomes. The polysaccharides (5, 10 and 25 microg ml(-1)) also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity. The most potent inhibitors of cytochrome P450 isozymes and ornithine decarboxylase activities were the polysaccharides from soybeans fermented with Agrocybe cylindracea. PMID:15000485

  1. Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

    PubMed

    Feder, Daniel; Hussein, Waleed M; Clayton, Daniel J; Kan, Meng-Wei; Schenk, Gerhard; McGeary, Ross P; Guddat, Luke W

    2012-11-01

    Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

  2. Binding of α,α-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design1

    PubMed Central

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W.

    2011-01-01

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design, and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2- methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase. PMID:21728378

  3. Physical Nature of Fatty Acid Amide Hydrolase Interactions with Its Inhibitors: Testing a Simple Nonempirical Scoring Model.

    PubMed

    Giedroyć-Piasecka, Wiktoria; Dyguda-Kazimierowicz, Edyta; Beker, Wiktor; Mor, Marco; Lodola, Alessio; Sokalski, W Andrzej

    2014-12-26

    Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the deactivating hydrolysis of fatty acid ethanolamide neuromodulators. FAAH inhibitors have gained considerable interest due to their possible application in the treatment of anxiety, inflammation, and pain. In the context of inhibitor design, the availability of reliable computational tools for predicting binding affinity is still a challenging task, and it is now well understood that empirical scoring functions have several limitations that in principle could be overcome by quantum mechanics. Herein, systematic ab initio analyses of FAAH interactions with a series of inhibitors belonging to the class of the N-alkylcarbamic acid aryl esters have been performed. In contrast to our earlier studies of other classes of enzyme-inhibitor complexes, reasonable correlation with experimental results required us to consider correlation effects along with electrostatic term. Therefore, the simplest comprehensive nonempirical model allowing for qualitative predictions of binding affinities for FAAH ligands consists of electrostatic multipole and second-order dispersion terms. Such a model has been validated against the relative stabilities of the benchmark S66 set of biomolecular complexes. As it does not involve parameters fitted to experimentally derived data, this model offers a unique opportunity for generally applicable inhibitor design and virtual screening. PMID:25420234

  4. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

    PubMed

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  5. In silico modification of suberoylanilide hydroxamic acid (SAHA) as potential inhibitor for class II histone deacetylase (HDAC)

    PubMed Central

    2011-01-01

    Background The cervical cancer is the second most prevalent cancer for the woman in the world. It is caused by the oncogenic human papilloma virus (HPV). The inhibition activity of histone deacetylase (HDAC) is a potential strategy for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is widely known as a low toxicity HDAC inhibitor. This research presents in silico SAHA modification by utilizing triazole, in order to obtain a better inhibitor. We conducted docking of the SAHA inhibitor and 12 modified versions to six class II HDAC enzymes, and then proceeded with drug scanning of each one of them. Results The docking results show that the 12 modified inhibitors have much better binding affinity and inhibition potential than SAHA. Based on drug scan analysis, six of the modified inhibitors have robust pharmacological attributes, as revealed by drug likeness, drug score, oral bioavailability, and toxicity levels. Conclusions The binding affinity, free energy and drug scan screening of the best inhibitors have shown that 1c and 2c modified inhibitors are the best ones to inhibit class II HDAC. PMID:22373132

  6. Recent advances in inhibitors of bacterial fatty acid synthesis type II (FASII) system enzymes as potential antibacterial agents.

    PubMed

    Wang, Yi; Ma, Shutao

    2013-10-01

    Bacterial infections are a constant and serious threat to human health. With the increase of multidrug resistance of clinically pathogenic bacteria, common antibiotic therapies have been less effective. Fatty acid synthesis type II (FASII) system enzymes are essential for bacterial membrane lipid biosynthesis and represent increasingly promising targets for the discovery of antibacterial agents with new mechanisms of action. This review highlights recent advances in inhibitors of bacterial FASII as potential antibacterial agents, paying special attention to the activities, mechanisms, and structure-activity relationships of those inhibitors that mainly target β-ketoacyl-ACP synthase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase. Although inhibitors with low nanomolar and selective activity against various bacterial FASII have entered clinical trials, further research is needed to expand upon both available and yet unknown scaffolds to identify new FASII inhibitors that may have antibacterial potential, particularly against resistant bacterial strains.

  7. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

    PubMed

    Vuille-dit-Bille, Raphael N; Camargo, Simone M; Emmenegger, Luca; Sasse, Tom; Kummer, Eva; Jando, Julia; Hamie, Qeumars M; Meier, Chantal F; Hunziker, Schirin; Forras-Kaufmann, Zsofia; Kuyumcu, Sena; Fox, Mark; Schwizer, Werner; Fried, Michael; Lindenmeyer, Maja; Götze, Oliver; Verrey, François

    2015-04-01

    Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

  8. Secretion of high-mannose-type alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase I inhibitor 1-deoxymannojirimycin.

    PubMed

    Gross, V; Steube, K; Tran-Thi, T A; McDowell, W; Schwarz, R T; Decker, K; Gerok, W; Heinrich, P C

    1985-07-01

    Two different forms of alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5-h labeling period with [35S]methionine the high-mannose-type precursor of alpha 1-proteinase inhibitor (Mr 49000) and alpha 1-acid glycoprotein (Mr 39 000) and the mature-complex-type alpha 1-proteinase inhibitor (Mr 54 000) and alpha 1-acid glycoprotein (Mr 43 000-60 000) could be immunoprecipitated from the cells, but only the complex-type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1-deoxymannojirimycin at a concentration of 4 mM, the 49 000-Mr form of alpha 1-proteinase inhibitor and the 39 000-Mr form of alpha 1-acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of alpha 1-proteinase inhibitor nor that of alpha 1-acid glycoprotein was impaired by 1-deoxymannojirimycin. While alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by hepatocytes treated with 4 mM 1-deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose-labeled oligosaccharides of alpha 1-proteinase inhibitor, secreted by 1-deoxymannojirimycin-treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio-Gel P-4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1-Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N-glycosylation of alpha 1-proteinase inhibitor or alpha 1-acid glycoprotein. PMID:3160588

  9. The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin.

    PubMed Central

    Brickman, T J; Armstrong, S K

    1996-01-01

    Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp. PMID:8550442

  10. A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening.

    PubMed

    Xu, Yongqian; Li, Benhao; Xiao, Liangliang; Ouyang, Jia; Sun, Shiguo; Pang, Yi

    2014-08-14

    A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay. PMID:24957006

  11. Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid.

    PubMed

    Beffagna, Nicoletta; Riva, Marzia Alessandra

    2011-06-01

    The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC-treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC-induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H+ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H+-ATPase; (2) treatment with 10 µM erythrosine B (EB) early and totally inhibits net H+ and K+ fluxes across the PM, indicative of the H+ pump activity; nevertheless, also in these conditions a huge FC-induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC-induced PM H+-ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂C₂-soluble fraction extracted from the incubation medium by sequential acid-base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC-induced catalase inhibition belongs to the organic acid class.

  12. Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1*

    PubMed Central

    Fagan, Rebecca L.; Cryderman, Diane E.; Kopelovich, Levy; Wallrath, Lori L.; Brenner, Charles

    2013-01-01

    Methylation of cytosines in CpG dinucleotides is the predominant epigenetic mark on vertebrate DNA. DNA methylation is associated with transcriptional repression. The pattern of DNA methylation changes during development and with disease. Human DNA methyltransferase 1 (Dnmt1), a 1616-amino acid multidomain enzyme, is essential for maintenance of DNA methylation in proliferating cells and is considered an important cancer drug target. Using a fluorogenic, endonuclease-coupled DNA methylation assay with an activated form of Dnmt1 engineered to lack the replication foci targeting sequence domain, we discovered that laccaic acid A (LCA), a highly substituted anthraquinone natural product, is a direct inhibitor with a 310 nm Ki. LCA is competitive with the DNA substrate in in vitro methylation assays and alters the expression of methylated genes in MCF-7 breast cancer cells synergistically with 5-aza-2′-deoxycytidine. LCA represents a novel class of Dnmt-targeted molecular probes, with biochemical properties that allow it to distinguish between non DNA-bound and DNA-bound Dnmt1. PMID:23839987

  13. Structural studies on sweet taste inhibitors: lactisole, DL-2(4-methoxyphenoxy)-propanoic acid

    NASA Astrophysics Data System (ADS)

    Matholouthi, M.; Angiboust, J. F.; Kacurakova, M.; Hooft, R. W. W.; Kanters, J. A.; Kroon, J.

    1994-09-01

    Lactisole, DL-2-(4-methoxyphenoxy)-propanoic acid (HPMP) has the formula C 10O 4H 12, Mr = 196.20, and is monoclinic, C2/c. a = 34.944(5), b = 5.2146(14), c = 11.201(2) Å, β = 101.495(13)°, V = 2000.1(7) Å 3, Z = 8, Dx = 1.3031(5) mg m -3, λ(Mo Kα) = 0.71073 Å, μ = 0.9 cm -1, F(000) = 832, R = 0.0392 for 1468 unique observed diffractometer data ( I ⩾ 2.5σ( I)). In the molecule two planar fragments, the acetic acid group and the phenyl ring, are almost perpendicular (interplanar angle 80.4(1)°). The crystal structure is characterized by cyclic dimers formed by hydrogen bonds between carboxyl groups across centers of inversion. The sodium salt of lactisole, NaPMP, is also a selective inhibitor of the sweetness of sucrose and was studied in aqueous solution in order to elucidate the mechanism of sweet taste inhibition. Solution properties, FT-IR spectra and the effect of NaPMP on the structure of water as determined by Raman spectra in the region of the OH stretching vibration were investigated. The hydrophobicity of NaPMP together with the steric hindrance caused by this molecule at the entrance of the sweet taste receptor site are probably at the origin of its inhibitory effect.

  14. Substituent effects of cis-cinnamic acid analogues as plant growh inhibitors.

    PubMed

    Nishikawa, Keisuke; Fukuda, Hiroshi; Abe, Masato; Nakanishi, Kazunari; Taniguchi, Tomoya; Nomura, Takashi; Yamaguchi, Chihiro; Hiradate, Syuntaro; Fujii, Yoshiharu; Okuda, Katsuhiro; Shindo, Mitsuru

    2013-12-01

    1-O-cis-Cinnamoyl-β-D-glucopyranose is one of the most potent allelochemicals that has been isolated from Spiraea thunbergii Sieb by Hiradate et al. It derives its strong inhibitory activity from cis-cinnamic acid (cis-CA), which is crucial for phytotoxicity. By preparing and assaying a series of cis-CA analogues, it was previously found that the key features of cis-CA for lettuce root growth inhibition are a phenyl ring, cis-configuration of the alkene moiety, and carboxylic acid. On the basis of a structure-activity relationship study, the substituent effects on the aromatic ring of cis-CA were examined by systematic synthesis and the lettuce root growth inhibition assay of a series of cis-CA analogues having substituents on the aromatic ring. While ortho- and para-substituted analogues exhibited low potency in most cases, meta-substitution was not critical for potency, and analogues having a hydrophobic and sterically small substituent were more likely to be potent. Finally, several cis-CA analogues were found to be more potent root growth inhibitors than cis-CA.

  15. Proton pump inhibitors--their pharmacological impact on the clinical management of acid-related disorders.

    PubMed

    Klotz, Ulrich

    2009-01-01

    Acid secretion or intragastric pH play a very important role in the pathophysiology of acid-related disorders such as peptic ulcer (PU), gastrooesophageal reflux disease (GERD) or nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal lesions. Proton pump inhibitors (PPIs) represent the most potent/effective antisecretory drugs for these indications. For the selection among the various agents (omeprazole/esomeprazole (CAS 73590-58-6/119141-88-7), pantoprazole (CAS 102625-70-7), lansoprazole (CAS103577-45-3), rabeprazole (CAS 117976-83-3)) some features of their pharmacokinetic (PK) and pharmacodynamic (PD) properties should be considered as the clinical outcome depends on systemic drug exposure (PK) and elevation of intragastric pH about certain threshold levels (PD). The present review updates PK, PD and clinical data to provide some guidance between the PPIs which differ somewhat in their metabolic pattern and drug interaction potential. Based on 24-h intragastric pH assessments the relative potencies of the PPIs compared to omeprazole were in healthy volunteers (in GERD patients): 0.42 (0.59), 1.0 (0.8), 1.0 (1.0), 1.25 (1.25) and 2.0 (1.4) for pantoprazole, lansoprazole, omeprazole, esomeprazole and rabeprazole, respectively. In general, the clinical benefits of PPI are well documented but some patients can be regarded as non-responders and thus represent a challenge for future clinical research. PMID:19634508

  16. Treatment with the hyaluronic Acid synthesis inhibitor 4-methylumbelliferone suppresses LPS-induced lung inflammation.

    PubMed

    McKallip, Robert J; Ban, Hao; Uchakina, Olga N

    2015-01-01

    Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS.

  17. Discovery of alpha,gamma-diketo acids as potent selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase.

    PubMed

    Summa, Vincenzo; Petrocchi, Alessia; Pace, Paola; Matassa, Victor G; De Francesco, Raffaele; Altamura, Sergio; Tomei, Licia; Koch, Uwe; Neuner, Philippe

    2004-01-01

    alpha,gamma-Diketo acids (DKA) were discovered from screening as selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase. The diketo acid moiety proved essential for activity, while substitution on the gamma position was necessary for selectivity and potency. Optimization led to the identification of a DKA inhibitor of NS5b polymerase with IC(50) = 45 nM, one of the most potent HCV NS5b polymerase inhibitors reported.

  18. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    PubMed Central

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-01-01

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present. PMID:8660289

  19. Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

    PubMed

    Vannice, John C; Skaff, D Andrew; Keightley, Andrew; Addo, James K; Wyckoff, Gerald J; Miziorko, Henry M

    2014-03-01

    Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase. PMID:24375100

  20. Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

    PubMed

    Aizawa, S; Miyasawa-Hori, H; Nakajo, K; Washio, J; Mayanagi, H; Fukumoto, S; Takahashi, N

    2009-01-01

    This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity. PMID:19136828

  1. Analogs of cinnamic acid benzyl amide as nonclassical inhibitors of activated JAK2 kinase.

    PubMed

    Mielecki, Marcin; Milner-Krawczyk, Małgorzata; Grzelak, Krystyna; Mielecki, Damian; Krzysko, Krystiana A; Lesyng, Bogdan; Priebe, Waldemar

    2014-01-01

    Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstratecompetitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC₅₀] = 14.8 µM), WP1130 (IC₅₀ = 3.8 µM), and WP1702 (IC₅₀ = 2.9 µM) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects.

  2. Acute Neurologic Disorder from an Inhibitor of Fatty Acid Amide Hydrolase.

    PubMed

    Kerbrat, Anne; Ferré, Jean-Christophe; Fillatre, Pierre; Ronzière, Thomas; Vannier, Stéphane; Carsin-Nicol, Béatrice; Lavoué, Sylvain; Vérin, Marc; Gauvrit, Jean-Yves; Le Tulzo, Yves; Edan, Gilles

    2016-11-01

    Background A decrease in fatty acid amide hydrolase (FAAH) activity increases the levels of endogenous analogues of cannabinoids, or endocannabinoids. FAAH inhibitors have shown analgesic and antiinflammatory activity in animal models, and some have been tested in phase 1 and 2 studies. In a phase 1 study, BIA 10-2474, an orally administered reversible FAAH inhibitor, was given to healthy volunteers to assess safety. Methods Single doses (0.25 to 100 mg) and repeated oral doses (2.5 to 20 mg for 10 days) of BIA 10-2474 had been administered to 84 healthy volunteers in sequential cohorts; no severe adverse events had been reported. Another cohort of participants was then assigned to placebo (2 participants) or 50 mg of BIA 10-2474 per day (6 participants). This report focuses on neurologic adverse events in participants in this final cohort. A total of 4 of the 6 participants who received active treatment consented to have their clinical and radiologic data included in this report. Results An acute and rapidly progressive neurologic syndrome developed in three of the four participants starting on the fifth day of drug administration. The main clinical features were headache, a cerebellar syndrome, memory impairment, and altered consciousness. Magnetic resonance imaging showed bilateral and symmetric cerebral lesions, including microhemorrhages and hyperintensities on fluid-attenuated inversion recovery and diffusion-weighted imaging sequences predominantly involving the pons and hippocampi. One patient became brain dead; the condition of two patients subsequently improved, but one patient had residual memory impairment, and the other patient had a residual cerebellar syndrome. One patient remained asymptomatic. Conclusions An unanticipated severe neurologic disorder occurred after ingestion of BIA 10-2474 at the highest dose level used in a phase 1 trial. The underlying mechanism of this toxic cerebral syndrome remains unknown.

  3. Perturbation of the Monomer-Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer

    SciTech Connect

    Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J.

    2014-08-14

    The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

  4. The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

    PubMed

    Negreiros, A N; Carvalho, M M; Xavier Filho, J; Blanco-Labra, A; Shewry, P R; Richardson, M

    1991-01-01

    The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin. PMID:1367792

  5. Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme: activity optimisation with conformationally rigid analogues of D-glutamic acid.

    PubMed

    Sosič, Izidor; Barreteau, Hélène; Simčič, Mihael; Sink, Roman; Cesar, Jožko; Zega, Anamarija; Grdadolnik, Simona Golič; Contreras-Martel, Carlos; Dessen, Andréa; Amoroso, Ana; Joris, Bernard; Blanot, Didier; Gobec, Stanislav

    2011-07-01

    D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities. PMID:21524830

  6. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  7. Keto-isovalerate decarboxylase enzymes and methods of use thereof

    DOEpatents

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian

    2016-01-19

    Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

  8. Multicomponent click synthesis of new 1,2,3-triazole derivatives of pyrimidine nucleobases: promising acidic corrosion inhibitors for steel.

    PubMed

    González-Olvera, Rodrigo; Espinoza-Vázquez, Araceli; Negrón-Silva, Guillermo E; Palomar-Pardavé, Manuel E; Romero-Romo, Mario A; Santillan, Rosa

    2013-12-06

    A series of new mono-1,2,3-triazole derivatives of pyrimidine nucleobases were synthesized by one-pot copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between N-1-propargyluracil and thymine, sodium azide and several benzyl halides. The desired heterocyclic compounds were obtained in good yields and characterized by NMR, IR, and high resolution mass spectrometry. These compounds were investigated as corrosion inhibitors for steel in 1 M HCl solution, using electrochemical impedance spectroscopy (EIS) technique. The results indicate that these heterocyclic compounds are promising acidic corrosion inhibitors for steel.

  9. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    SciTech Connect

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  10. Fatty Acid Transport Protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    PubMed Central

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC50 8–11μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC50 58μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13C-oleate demonstrating its potential as a therapeutic agent. PMID:26284975

  11. Examination of the Addictive and Behavioral Properties of Fatty Acid-Binding Protein Inhibitor SBFI26

    PubMed Central

    Thanos, Panayotis K.; Clavin, Brendan H.; Hamilton, John; O’Rourke, Joseph R.; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested. PMID:27092087

  12. Evaluation of the radiolabeled boronic acid-based FAP inhibitor MIP-1232 for atherosclerotic plaque imaging.

    PubMed

    Meletta, Romana; Müller Herde, Adrienne; Chiotellis, Aristeidis; Isa, Malsor; Rancic, Zoran; Borel, Nicole; Ametamey, Simon M; Krämer, Stefanie D; Schibli, Roger

    2015-01-01

    Research towards the non-invasive imaging of atherosclerotic plaques is of high clinical priority as early recognition of vulnerable plaques may reduce the incidence of cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed as inflammation-induced protease involved in the process of plaque vulnerability. In this study, FAP mRNA and protein levels were investigated by quantitative polymerase chain reaction and immunohistochemistry, respectively, in human endarterectomized carotid plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and radiolabeled with iodine-125. The potential of this radiotracer to image plaques was evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed with a xenograft with high and one with low FAP level, grown in mice. Target expression analyses revealed a moderately higher protein level in atherosclerotic plaques than normal arteries correlating with plaque vulnerability. No difference in expression was determined on mRNA level. The radiotracer was successfully produced and accumulated strongly in the FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging. PMID:25633335

  13. Salubrinal, ER stress inhibitor, attenuates kainic acid-induced hippocampal cell death.

    PubMed

    Kim, Jung Soo; Heo, Rok Won; Kim, Hwajin; Yi, Chin-Ok; Shin, Hyun Joo; Han, Jong Woo; Roh, Gu Seob

    2014-10-01

    Kainic acid (KA)-induced neuronal death is closely linked to endoplasmic reticulum (ER) and mitochondrial dysfunction. Parkin is an ubiquitin E3 ligase that mediates the ubiquitination of the Bcl-2 family of proteins and its mutations are associated with neuronal apoptosis in neurodegenerative diseases. We investigated the effect of salubrinal, an ER stress inhibitor, on the regulation of ER stress and mitochondrial apoptosis induced by KA, in particular, by controlling parkin expression. We showed that salubrinal significantly reduced seizure activity and increased survival rates of mice with KA-induced seizures. We found that salubrinal protected neurons against apoptotic death by reducing expression of mitochondrial apoptotic factors and elF2α-ATF4-CHOP signaling proteins. Interestingly, we showed that salubrinal decreased the KA-induced parkin expression and inhibited parkin translocation to mitochondria, which suggests that parkin may regulate a cross-talk between ER and mitochondria. Collectively, inhibition of ER stress attenuates mitochondrial apoptotic and ER stress pathways and controls parkin-mediated neuronal death following KA-induced seizures. PMID:24728926

  14. A lysophosphatidic acid analogue is revealed as a potent inhibitor of phosphatidylcholine synthesis, inducing apoptosis.

    PubMed Central

    Gueguen, Geneviéve; Granci, Virginie; Rogalle, Pierre; Briand-Mésange, Fabienne; Wilson, Michéle; Klaébé, Alain; Tercé, François; Chap, Hugues; Salles, Jean-Pierre; Simon, Marie-Françoise; Gaits, Frédérique

    2002-01-01

    A previous study demonstrated that cross-desensitization experiments performed with the lysophosphatidic acid (LPA) analogues (R)- and (S)-N-palmitoyl-norleucinol 1-phosphate (PNPAs) inhibited LPA-induced platelet aggregation without any stereospecificity. Here we report opposite biological effects of the two enantiomers on mitogenesis of IMR-90 fibroblasts in relation to their respective metabolism. (R)PNPA was proliferative, while (S)PNPA induced apoptosis by specifically inhibiting phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by cholinephosphotransferase. This effect was not direct but required dephosphorylation of PNPAs by ecto-lipid phosphate phosphatase before cellular uptake of the generated N-palmitoyl-norleucinols (PNOHs). Inhibition of cholinephosphotransferase by the derivative (S)PNOH was confirmed by an in vitro assay. (S)PNPA proapoptotic effects led us to clarify the mechanism linking cholinephosphotransferase inhibition to apoptosis. Three proapoptotic responses were observed: the activation of caspase-3, the production of ceramides from newly synthesized pools (as demonstrated by the inhibitor Fumonisin B1) and finally the activation of stress-activated protein kinase, p38 and c-Jun N-terminal kinases 1/2, as a result of ceramide increase. Thus our data demonstrate that synthetic analogues of LPA might display stereospecific effects leading to apoptosis independently of classical LPA-activated pathways. PMID:12197836

  15. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, attenuates postoperative cognitive dysfunction in aging mice

    PubMed Central

    Jia, Min; Liu, Wen-Xue; Sun, He-Liang; Chang, Yan-Qing; Yang, Jiao-Jiao; Ji, Mu-Huo; Yang, Jian-Jun; Feng, Chen-Zhuo

    2015-01-01

    Postoperative cognitive dysfunction (POCD) is a recognized clinical entity characterized with cognitive deficits after anesthesia and surgery, especially in aged patients. Previous studies have shown that histone acetylation plays a key role in hippocampal synaptic plasticity and memory formation. However, its role in POCD remains to be determined. Here, we show that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, attenuates POCD in aging Mice. After exposed to the laparotomy, a surgical procedure involving an incision into abdominal walls to examine the abdominal organs, 16- but not 3-month old male C57BL/6 mice developed obvious cognitive impairments in the test of long-term contextual fear conditioning. Intracerebroventricular (i.c.v.) injection of SAHA at the dose of (20 μg/2 μl) 3 h before and daily after the laparotomy restored the laparotomy-induced reduction of hippocampal acetyl-H3 and acetyl-H4 levels and significantly attenuated the hippocampus-dependent long-term memory (LTM) impairments in 16-month old mice. SAHA also reduced the expression of cleaved caspase-3, inducible nitric oxide synthase (iNOS) and N-methyl-D-aspartate (NMDA) receptor-calcium/calmodulin dependent kinase II (CaMKII) pathway, and increased the expression of brain-derived neurotrophic factor (BDNF), synapsin 1, and postsynaptic density 95 (PSD95). Taken together, our data suggest that the decrease of histone acetylation contributes to POCD and may serve as a target to improve the neurological outcome of POCD. PMID:26441515

  16. Characterization of a second ornithine decarboxylase isolated from Morganella morganii.

    PubMed

    De Las Rivas, Blanca; González, Ramón; Landete, José María; Muñoz, Rosario

    2008-03-01

    The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

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

    PubMed

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

    1999-11-01

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

  18. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

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

    PubMed

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

    1999-11-01

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

  20. Mechanism for release of arachidonic acid during guinea pig platelet aggregation: a role for the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.

    1986-01-01

    The mechanism of the release of arachidonic acid from phospholipids after the stimulation of guinea pig platelets with collagen, thrombin and platelet activating factor (PAF) was studied. RHC 80267, a diacylglycerol lipase inhibitor, and indomethacin, a cyclooxygenase inhibitor, were used. Various in vitro assays for enzymes involved in arachidonic acid release and metabolism were conducted. Platelet aggregation and simultaneous release of ADP from platelets were monitored using a Chrono-log Lumiaggregometer. Platelets were labeled with (/sup 14/C)arachidonic acid to facilitate sensitive determination of small changes in platelet phospholipids during platelet aggregation. In the present investigation it is shown that collagen, thrombin and PAF increased phospholipase C activity. It was also discovered that cyclooxygenase products were responsible for further stimulation (a positive feed-back) of phospholipase C activity, while diacylglycerol provided a negative feed-back control over receptor-stimulated phospholipase C activity and inhibited ADP release. The guinea pig platelet is an ideal model to study phospholipase C-diacylglycerol lipase pathway for the release of arachidonic acid from platelet phospholipids because it does not have any phospholipase A/sub 2/ activity. It was observed that cyclooxygenase products were responsible for collagen-induced guinea pig platelet aggregation. Indomethacin completely inhibited collagen-induced platelet aggregation, was less effective against thrombin, and had no effect on PAF-induced platelet aggregation. On the other hand, RHC 80267 was a powerful inhibitor of aggregation and ADP release induced by all three of these potent aggregating agents.

  1. Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

    PubMed Central

    Norman, Shirley M.; Poling, Stephen M.; Maier, Vincent P.; Orme, Edward D.

    1983-01-01

    The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds. PMID:16662775

  2. Design and synthesis of inhibitors incorporating beta -amino acids of metalloendopeptidase EC 3.4.24.15.

    PubMed

    Steer, D L; Lew, R A; Perlmutter, P; Smith, A I; Aguilar, M I

    2000-09-01

    Endopeptidase EC 3.4.24.15 (EP 24.15) is a thermolysin-like metalloendopeptidase which is expressed widely throughout the body, with the highest concentrations in the brain, pituitary and testis. While the precise role of EP 24.15 remains unknown, it is thought to participate in the regulated metabolism of a number of specific neuropeptides. Of the limited number of inhibitors described for EP 24.15, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but is unstable in vivo due to its cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). The cpp-Ala-Ala N-terminal product of this cleavage is a potent inhibitor of angiotensin converting enzyme, which further limits the use of CFP in vivo. To generate specific inhibitors of EP 24.15 that are resistant to in vivo proteolysis by EP 24.11, beta-amino acids have been incorporated into the structure of CFP. We have prepared racemic mixtures of beta-amino acids containing proteogenic side chains, which are 9-fluorenylmethoxycarbonyl (Fmoc)-protected, and several analogues of CFP containing beta-amino acids have been synthesized by solid phase peptide synthesis. The results of stability and inhibitory studies of these new analogues show that the incorporation of beta-amino acids adjacent to the scissile bond can indeed stabilize the peptides against cleavage by EP 24.11 and still inhibit EP 24.15. The results obtained in these studies demonstrate the potential of these amino acids in the synthesis of peptidomimetics and in the design of new stable and specific therapeutics. PMID:11016884

  3. Ornithine decarboxylase and S-adenosyl methionine decarboxylase in skin fibroblasts of normal and cystic fibrosis patients.

    PubMed

    Buehler, B; Wright, R; Schott, S; Darby, B; Rennert, O M

    1977-03-01

    The key enzymes in the synthesis of the naturally occurring polyamines, ornithine decarboxylase (ODC) and S-adenosyl methionine (SAM) decarboxylase, were investigated during cell growth and aging in fibroblast cultures from normal patients and patients with cystic fibrosis. A linear correlation between increased S-adenosyl methionine activity and putrescine concentration was apparent in all cell lines. A putrescine concentration of 0.8 mM was optimal for enhancement of SAM decarboxylase activity. The passage number of the cell line correlated inversely with maximal putrescine-stimulated SAM decarboxylase activity, earlier passage numbers having the highest specific activity (Fig. 1). No significant differences in basal or putrescine-stimulated SAM decarboxylase activity were noted between normal fibroblast cultures and cells from patients with cystic fibrosis (Fig. 2). SAM decarboxylase activity increased as the cell lines approached confluence. Activity was lowest during exponential growth (Fig. 3). ODC activity was increased during early exponential growth and fell as cells reached confluence (Fig. 4). No differences in ODC activity and putrescine inhibition between the normal and cystic fibrosis cell cultures at equivalent points of exponential growth were noted.

  4. Application of computational methods to the design of fatty acid amide hydrolase (FAAH) inhibitors based on a carbamic template structure.

    PubMed

    Lodola, Alessio; Rivara, Silvia; Mor, Marco

    2011-01-01

    Computer-aided approaches are widely used in modern medicinal chemistry to improve the efficiency of the discovery phase. Fatty acid amide hydrolase (FAAH) is a key component of the endocannabinoid system and a potential drug target for several therapeutic applications. During the past decade, different chemical classes of inhibitors, with different mechanisms of action, had been developed. Among them, alkyl carbamic acid biphenyl-3-yl esters represent a prototypical class of active site-directed inhibitors, which allowed detailed pharmacological characterization of FAAH inhibition. Both ligand- and structure-based drug design approaches have been applied to rationalize structure-activity relationships and to drive the optimization of the inhibitory potency for this class of compounds. In this chapter, we review our contribution to the discovery and optimization of therapeutically promising FAAH inhibitors, based on a carbamic template structure, which block FAAH in an irreversible manner exerting analgesic, anti-inflammatory and anxiolytic effects in animal models. The peculiar catalytic mechanism of FAAH, and the covalent interaction with carbamate-based inhibitors, prompted the application of different computer-aided tools, ranging from ligand-based approaches to docking procedures and quantum mechanics/molecular mechanics (QM/MM) hybrid techniques. Latest advancements in the field are also reported.

  5. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  6. Some aromatic hydrazone derivatives as inhibitors for the corrosion of C-steel in phosphoric acid solution.

    PubMed

    Fouda, Abd El-Aziz S; Al-Sarawy, Ahmed A; Radwan, Mohamed S

    2006-01-01

    The effect of furfural benzoylhydrazone and its derivatives (I-VII) as corrosion inhibitors for C-steel in 1M phosphoric acid solution has been studied by weight-loss and galvanostatic polarization techniques. A significant decrease in the corrosion rate of C-steel was observed in the presence of the investigated inhibitors. This study revealed that, the inhibition efficiency increases with increasing the inhibitor concentration, and the addition of iodide ions enhances it to a considerable extent. The effect of temperature on the inhibition efficiency of these compounds was studied using weight-loss method. Activation energy (E(a)*) and other thermodynamic parameters for the corrosion process were calculated and discussed. The galvanostatic polarization data indicated that, the inhibitors were of mixed-type, but the cathode is more polarized than the anode. The adsorption of these compounds on C-steel surface has been found to obey Frumkin's adsorption isotherm. The mechanism of inhibition was discussed in the light of the chemical structure of the undertaken inhibitors.

  7. Valoniopsis pachynema Extract as a Green Inhibitor for Corrosion of Brass in 0.1 N Phosphoric Acid Solution

    NASA Astrophysics Data System (ADS)

    Selva Kumar, R.; Chandrasekaran, V.

    2016-04-01

    The effect of marine alga Valoniopsis pachynema extract on corrosion inhibition of brass in phosphoric acid was investigated by weight-loss method, potentiodynamic polarization, and electrochemical impedance spectroscopy studies. The inhibition efficiency is found to increase with increasing concentration of extract and decreases with rise in temperature. The activation energy, thermodynamic parameters (free energy, enthalpy, and entropy change) and kinetic parameters (rate constant and half-life) for inhibition process were calculated. These thermodynamic and kinetic parameters indicate a strong interaction between the inhibitor and the brass surface. The inhibition is assumed to occur via adsorption of inhibitor molecules on brass surface, which obeys Temkin adsorption isotherm. The adsorption of inhibitor on the brass surface is exothermic, physical, and spontaneous, and follows first-order kinetics. The polarization measurements showed that the inhibitor behaves as a mixed type inhibitor and the higher inhibition surface coverage on the brass was predicted. Inhibition efficiency values were found to show good trend with weight-loss method, potentiodynamic polarization, and electrochemical impedance spectroscopy studies. Surface study techniques (FT-IR and SEM) were carried out to ascertain the inhibitive nature of the algal extract on the brass surface.

  8. Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase.

    PubMed

    Taylor, Nicolas L; Day, David A; Millar, A Harvey

    2002-11-01

    A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with a K(0.5) of 30 microm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria.

  9. Analogs of the antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Pugliese, Kaitlin M; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2012-11-01

    Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.

  10. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    PubMed

    Björklund, H V; Johansson, T R; Rinne, A

    1997-07-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor. PMID:9188644

  11. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus 1

    PubMed Central

    Legaz, María Estrella; Vicente, Carlos

    1983-01-01

    Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID:16662821

  12. Properties of oxaloacetate decarboxylase from Veillonella parvula.

    PubMed Central

    Ng, S K; Wong, M; Hamilton, I R

    1982-01-01

    Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe. PMID:7076619

  13. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  14. From dihydroxypyrimidine carboxylic acids to carboxamide HIV-1 integrase inhibitors: SAR around the amide moiety.

    PubMed

    Petrocchi, Alessia; Koch, Uwe; Matassa, Victor G; Pacini, Barbara; Stillmock, Kara A; Summa, Vincenzo

    2007-01-15

    4,5-Dihyroxypyrimidine carboxamides, which evolved from a related series of HCV NS5b polymerase inhibitors, have been optimized to provide selective HIV integrase strand transfer inhibitors. Extensive SAR around the benzylamide moiety led to the identification of the p-fluorobenzylamide as optimal in the enzymatic assay.

  15. Ferulic acid, an efficient inhibitor of type B trichothecene biosynthesis and Tri gene expression in Fusarium liquid cultures.

    PubMed

    Boutigny, Anne-Laure; Barreau, Christian; Atanasova-Penichon, Vessela; Verdal-Bonnin, Marie-Noëlle; Pinson-Gadais, Laëtitia; Richard-Forget, Florence

    2009-01-01

    The effect of ferulic acid, the most abundant phenolic acid in wheat bran, was studied in vitro on type B trichothecene biosynthesis by Fusarium. It was demonstrated that ferulic acid is an efficient inhibitor of mycotoxin production by all strains of Fusarium tested, including different chemotypes and species. To analyse the mechanism of toxin biosynthesis inhibition by ferulic acid, expression of representative Tri genes, involved in the trichothecene biosynthesis pathway, was monitored by real-time RT-PCR. A decrease in the level of Tri gene expression was measured, suggesting that inhibition of toxin synthesis by ferulic acid could be regulated at the transcriptional level. Moreover, toxin production was shown to be reduced proportionally to the initial amount of ferulic acid added in the culture medium. Addition of ferulic acid either at the spore germination step or to a mycelial culture resulted in the same final inhibitory effect on mycotoxin accumulation. A cumulative inhibitory effect on trichothecene biosynthesis was even observed with successive supplementation of ferulic acid. Ferulic acid, which content varies among wheat varieties, could then play an important role in modulating trichothecene biosynthesis by Fusarium in some wheat varieties.

  16. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  17. Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.

    PubMed

    Varasi, Mario; Thaler, Florian; Abate, Agnese; Bigogno, Chiara; Boggio, Roberto; Carenzi, Giacomo; Cataudella, Tiziana; Dal Zuffo, Roberto; Fulco, Maria Carmela; Rozio, Marco Giulio; Mai, Antonello; Dondio, Giulio; Minucci, Saverio; Mercurio, Ciro

    2011-04-28

    New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

  18. Electrochemical Study of AISI C1018 Steel in Methanesulfonic Acid Containing an Acetylenic Alcohol-Based Corrosion Inhibitor Formulation.

    PubMed

    Finšgar, Matjaž; Jackson, Jennifer

    2016-10-01

    In this work, the electrochemical potentiodynamic behavior of AISI C1018 lower-grade steel material was investigated in 20 wt.% methanesulfonic acid (MSA) solutions with or without different components to design corrosion inhibitor formulations based on acetylenic alcohol, cinnamaldehyde, 1-dodecylpyridinium chloride, and methanol. MSA has recently been considered as a new potential acid to be used in the matrix stimulation procedure and in well cleaning. It is demonstrated that AISI C1018 steel MSA needs to be inhibited. Inhibition type is determined for single components as well as for formulations.

  19. Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

    PubMed Central

    Schrader, Florian C.; Glinca, Serghei; Sattler, Julia M.; Dahse, Hans-Martin; Afanador, Gustavo A.; Prigge, Sean T.; Lanzer, Michael; Mueller, Ann-Kristin; Klebe, Gerhard; Schlitzer, Martin

    2013-01-01

    Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC50 values of 1.7 and 3.0 µm and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine. PMID:23341167

  20. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

    SciTech Connect

    Zhai, Yingying; Chen, Xi; Yu, Dehai; Li, Tao; Cui, Jiuwei; Wang, Guanjun; Hu, Ji-Fan; Li, Wei

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  1. Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Kondo, Yuki; Iwao, Takahiro; Yoshihashi, Sachimi; Mimori, Kayo; Ogihara, Ruri; Nagata, Kiyoshi; Kurose, Kouichi; Saito, Masayoshi; Niwa, Takuro; Suzuki, Takayoshi; Miyata, Naoki; Ohmori, Shigeru; Nakamura, Katsunori; Matsunaga, Tamihide

    2014-01-01

    In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. PMID:25084468

  2. Efficacy of phytic acid as an inhibitor of enzymatic and non-enzymatic browning in apple juice.

    PubMed

    Du, Yunjian; Dou, Siqi; Wu, Shengjun

    2012-11-15

    Browning decreases the commercial value of apple juice, and therefore colour preservation during processing and storage is the main objective of manufacturers. In this study, the efficacy of phytic acid as a browning inhibitor for use on apple juice was investigated. Browning of apple juice treated with phytic acid was monitored during processing and storage. 0.1 mM Phytic acid inhibited the polyphenol oxidase (PPO) from the apple juice by 99.2%. Consequently, the apple juice treated with phytic acid had significantly lower browning formation during processing and after 6 months of storage at room temperature compared with the control (p<0.05). Results indicate that this is a promising way to inhibit browning in apple juice.

  3. Comparison of amino acid sequences of the trypsin inhibitors from taro (Colocasia esculenta), giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis).

    PubMed

    Peng, L; Bradbury, J H; Hammer, B C; Shaw, D C

    1993-09-01

    The amino acid sequences of the trypsin inhibitors from taro Colocasia esculenta var. esculenta and giant swamp taro Cyrtosperma chamissonis have been determined and are compared with the protein sequence of the trypsin/chymotrypsin inhibitor from giant taro Alocasia macrorrhiza. Both inhibitors display polymorphism and there is evidence of two components in the giant swamp taro. The positional identity between the proteins is highest at 73-75% for the comparison of the giant taro (GT) with the polymorphic forms of the taro (T) inhibitors and lowest at 56-58% for the pairs of taro and giant swamp taro (GST) proteins. The comparisons show that the inhibitors from T and GT are more related to each other than to GST, which supports their taxonomic classification into different tribes. Location of the P1 site for the trypsin inhibitors of aroids is different from that of other Kunitz-type inhibitors and could be at Leu56.

  4. In silico Analysis for Predicting Fatty Acids of Black Cumin Oil as Inhibitors of P-Glycoprotein

    PubMed Central

    Ali, Babar; Jamal, Qazi Mohd. Sajid; Mir, Showkat R.; Shams, Saiba; Al-Wabel, Naser A.; Kamal, Mohammad A.

    2015-01-01

    Background: Black cumin oil is obtained from the seeds of Nigella sativa L. which belongs to family Ranunculaceae. The seed oil has been reported to possess antitumor, antioxidant, antibacterial, anti-inflammatory, hypoglycemic, central nervous system depressant, antioxidant, and immunostimulatory activities. These bioactivities have been attributed to the fixed oil, volatile oil, or their components. Seed oil consisted of 15 saturated fatty acids (17%) and 17 unsaturated fatty acids (82.9%). Long chain fatty acids and medium chain fatty acids have been reported to increase oral bioavailability of peptides, antibiotics, and other important therapeutic agents. In earlier studies, permeation enhancement and bioenhancement of drugs has been done with black cumin oil. Objective: In order to recognize the mechanism of binding of fatty acids to P-glycoprotein (P-gp), linoleic acid, oleic acid, margaric acid, cis-11, 14-eicosadienoic acid, and stearic acid were selected for in silico studies, which were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. Materials and Methods: Template search with BLAST and HHblits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence of P-gp from Rattus norvegicus. Results: The amount of energy needed by linoleic acid, oleic acid, eicosadienoic acid, margaric acid, and stearic acid to bind with P-gp were found to be − 10.60, −10.48, −9.95, −11.92, and − 10.37 kcal/mol, respectively. The obtained data support that all the selected fatty acids have contributed to inhibit P-gp activity thereby enhances the bioavailability of drugs. Conclusion: This study plays a significant role in finding hot spots in P-gp and may offer the further scope of designing potent and specific inhibitors of P-gp. SUMMARY Generation of 3D structure of fatty acid compounds from Black cumin oil and 3D homology modeling of Rat P

  5. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  6. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed Central

    Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.

    2016-01-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  7. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  8. The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase.

    PubMed

    Sievers, Annette; Wolfenden, Richard

    2005-02-01

    The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.

  9. Inhibitors of cyclooxygenase-2, but not cyclooxygenase-1 provide structural and functional protection against quinolinic acid-induced neurodegeneration.

    PubMed

    Salzberg-Brenhouse, Heather C; Chen, Er-Yun; Emerich, Dwaine F; Baldwin, Sam; Hogeland, Ken; Ranelli, Sharon; Lafreniere, Denise; Perdomo, Brigido; Novak, Leah; Kladis, Theodora; Fu, Karen; Basile, Anthony S; Kordower, Jeffrey H; Bartus, Raymond T

    2003-07-01

    Cyclooxygenases (COXs) are implicated in neurodegenerative processes associated with acute and chronic neurological diseases. Given the potential utility of COX inhibitors in treating these disorders, we examined the nonselective COX inhibitor flurbiprofen, the specific COX-1 inhibitor valeryl salicylate (VS), and the COX-2 inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS-398) for their abilities to protect striatal neurons against a quinolinic acid (QA)-induced excitotoxic lesion. Rats were administered COX inhibitors 10 min before a unilateral QA lesion of the striatum, and then tested 2 to 3 weeks later in a battery of motor tasks (bracing, placing, akinesia, and apomorphine-induced rotations). Lesion volume was assessed using immunohistochemical methods 1 month after lesioning. Orally administered flurbiprofen (50 mg) was highly neuroprotective, preserving 84 to 99% of motor performance (ED50 = 8.6-9.7 mg) while reducing lesion volume 75% (ED50 = 3.2 mg). The identities of the COX isoforms associated with QA-induced neurodegeneration were determined using VS and NS-398. Oral VS was ineffective in virtually all indices of functional neuroprotection. In contrast, oral NS-398 was highly effective, preserving approximately 83% of motor performance at2mg(ED50 = 0.1-0.4 mg), and reducing lesion volume 100% (ED50 = 0.4 mg). Similar results were obtained using inhaled flurbiprofen (2 mg), which preserved 88 to 100% of motor performance while reducing striatal lesion size 92%. These results demonstrate that COX-2 inhibition protects neurons from acute, excitotoxic neurodegeneration. Moreover, formulating a nonselective COX inhibitor into an inhalable preparation dramatically improves its potency in treating acute neuronal damage, a situation where the rapidity of drug delivery and onset of action is critical to clinical efficacy.

  10. Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

    PubMed Central

    Birecka, Helena; Birecki, Mieczyslaw; Cohen, Eric J.; Bitonti, Alan J.; McCann, Peter P.

    1988-01-01

    When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here—using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors—endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence—with relatively very high levels of these compounds—in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence. PMID:16665870

  11. Oxamic acid analogues as LDH-C4-specific competitive inhibitors.

    PubMed

    Rodríguez-Páez, Lorena; Chena-Taboada, Miguel Angel; Cabrera-Hernández, Arturo; Cordero-Martínez, Joaquín; Wong, Carlos

    2011-08-01

    We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.

  12. Frequent coexpression of the vesicular glutamate transporter 1 and 2 genes, as well as coexpression with genes for choline acetyltransferase or glutamic acid decarboxylase in neurons of rat brain.

    PubMed

    Danik, Marc; Cassoly, Estelle; Manseau, Frédéric; Sotty, Florence; Mouginot, Didier; Williams, Sylvain

    2005-08-15

    It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single-cell multiplex RT-PCR (sc-RT-mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT-PCR and quantitated by ribonuclease protection assay. VGLUT1 and -2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1-CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc-RT-mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for gamma-aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats. PMID:15983996

  13. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  14. A new dioxime corrosion inhibitor for the protection and conservation of copper: synthesis, characterization and evaluation in acidic chloride solution

    NASA Astrophysics Data System (ADS)

    Abu-Baker, Ahmad N.; Al-Qudah, Mahmoud A.

    2016-08-01

    This study aimed to investigate a new dioxime compound as a corrosion inhibitor for copper. The compound (4,6-dihydroxy benzene-1,3-dicarbaldehyde dioxime) was synthesized and characterized by nuclear magnetic resonance spectroscopy. Electrochemical impedance spectroscopy and potentiodynamic polarization measurements were used to compare the dioxime compound with benzotriazole for their effectiveness as corrosion inhibitors for copper in 0.1 M HCl solution. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to investigate the bonding mechanisms and morphological changes of the two inhibitors on the copper surface. The electrochemical techniques showed that the new dioxime compound was more effective than benzotriazole in inhibiting copper corrosion in the acidic chloride medium. The FTIR and SEM results indicated that the dioxime compound was able to coordinate with copper ions and formed a protective film on the copper surface. It was concluded that the new dioxime compound proved effectiveness to be used as a corrosion inhibitor for the protection and conservation of copper.

  15. Design and Synthesis of Potent N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA) Inhibitor as Anti-Inflammatory Compounds

    PubMed Central

    Chen, Ling; Zhu, Chenggang; Huang, Rui; Zheng, Xiao; Qiu, Yan; Fu, Jin

    2012-01-01

    N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme involved in biological deactivation of N-palmitoylethanolamide (PEA), which exerts anti-inflammatory and analgesic effects through the activation of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-α). To develop selective and potent NAAA inhibitors, we designed and synthesized a series of derivatives of 1-pentadecanyl-carbonyl pyrrolidine (compound 1), a general amidase inhibitor. Structure activity relationship (SAR) studies have identified a compound 16, 1-(2-Biphenyl-4-yl)ethyl-carbonyl pyrrolidine, which has shown the highest inhibition on NAAA activity (IC50 = 2.12±0.41 µM) and is characterized as a reversible and competitive NAAA inhibitor. Computational docking analysis and mutagenesis study revealed that compound 16 interacted with Asparagine 209 (Asn209) residue flanking the catalytic pocket of NAAA so as to block the substrate entrance. In vitro pharmacological studies demonstrated that compound 16 dose-dependently reduced mRNA expression levels of iNOS and IL-6, along with an increase of intracellular PEA levels, in mouse macrophages with lipopolysaccharides (LPS) induced inflammation. Our study discovered a novel NAAA inhibitor, compound 16, that could serve as a potential anti-inflammatory agent. PMID:22916199

  16. New coumarin derivative as an eco-friendly inhibitor of corrosion of mild steel in Acid medium.

    PubMed

    Al-Amiery, Ahmed A; Al-Majedy, Yasameen K; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

    2014-12-29

    The anticorrosion ability of a synthesized coumarin, namely 2-(coumarin-4-yloxy)acetohydrazide (EFCI), for mild steel (MS) in 1 M hydrochloric acid solution has been studied using a weight loss method. The effect of temperature on the corrosion rate was investigated, and some thermodynamic parameters were calculated. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. The IE value reaches 94.7% at the highest used concentration of the new eco-friendly inhibitor. The adsorption of inhibitor on MS surface was found to obey a Langmuir adsorption isotherm. Scanning electron microscopy (SEM) was performed on inhibited and uninhibited mild steel samples to characterize the surface. The Density Function theory (DFT) was employed for quantum-chemical calculations such as EHOMO (highest occupied molecular orbital energy), ELUMO (lowest unoccupied molecular orbital energy) and μ (dipole moment), and the obtained results were found to be consistent with the experimental findings. The synthesized inhibitor was characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies.

  17. Inhibiting the β-Lactamase of Mycobacterium tuberculosis (Mtb) with Novel Boronic Acid Transition-State Inhibitors (BATSIs).

    PubMed

    Kurz, Sebastian G; Hazra, Saugata; Bethel, Christopher R; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; Blanchard, John S; Bonomo, Robert A

    2015-06-12

    BlaC, the single chromosomally encoded β-lactamase of Mycobacterium tuberculosis, has been identified as a promising target for novel therapies that rely upon β-lactamase inhibition. Boronic acid transition-state inhibitors (BATSIs) are a class of β-lactamase inhibitors which permit rational inhibitor design by combinations of various R1 and R2 side chains. To explore the structural determinants of effective inhibition, we screened a panel of 25 BATSIs to explore key structure-function relationships. We identified a cefoperazone analogue, EC19, which displayed slow, time-dependent inhibition against BlaC with a potency similar to that of clavulanate (Ki* of 0.65 ± 0.05 μM). To further characterize the molecular basis of inhibition, we solved the crystallographic structure of the EC19-BlaC(N172A) complex and expanded our analysis to variant enzymes. The results of this structure-function analysis encourage the design of a novel class of β-lactamase inhibitors, BATSIs, to be used against Mycobacterium tuberculosis. PMID:27622739

  18. Fatty Acid-binding Proteins Transport N-Acylethanolamines to Nuclear Receptors and Are Targets of Endocannabinoid Transport Inhibitors*

    PubMed Central

    Kaczocha, Martin; Vivieca, Stephanie; Sun, Jing; Glaser, Sherrye T.; Deutsch, Dale G.

    2012-01-01

    N-Acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-14C]ethanolamide ([14C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter. PMID:22170058

  19. New coumarin derivative as an eco-friendly inhibitor of corrosion of mild steel in Acid medium.

    PubMed

    Al-Amiery, Ahmed A; Al-Majedy, Yasameen K; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

    2015-01-01

    The anticorrosion ability of a synthesized coumarin, namely 2-(coumarin-4-yloxy)acetohydrazide (EFCI), for mild steel (MS) in 1 M hydrochloric acid solution has been studied using a weight loss method. The effect of temperature on the corrosion rate was investigated, and some thermodynamic parameters were calculated. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. The IE value reaches 94.7% at the highest used concentration of the new eco-friendly inhibitor. The adsorption of inhibitor on MS surface was found to obey a Langmuir adsorption isotherm. Scanning electron microscopy (SEM) was performed on inhibited and uninhibited mild steel samples to characterize the surface. The Density Function theory (DFT) was employed for quantum-chemical calculations such as EHOMO (highest occupied molecular orbital energy), ELUMO (lowest unoccupied molecular orbital energy) and μ (dipole moment), and the obtained results were found to be consistent with the experimental findings. The synthesized inhibitor was characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies. PMID:25551187

  20. Synthesis and QSAR of Fatty Acid Amide Hydrolase Inhibitors: Modulation at the N-Portion of Biphenyl-3-yl Alkylcarbamates

    PubMed Central

    Mor, Marco; Lodola, Alessio; Rivara, Silvia; Vacondio, Federica; Duranti, Andrea; Tontini, Andrea; Sanchini, Silvano; Piersanti, Giovanni; Clapper, Jason R.; King, Alvin R.; Tarzia, Giorgio; Piomelli, Daniele

    2013-01-01

    Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (IC50, for FAAH = 63 nM) with a selected set of substituents of different size, shape, flexibility and lipophilicity. Docking experiments and Linear Interaction Energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the β-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3′-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors. PMID:18507372

  1. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci.

    PubMed

    Lee, Sang Ho; Wang, Hao; Labroli, Marc; Koseoglu, Sandra; Zuck, Paul; Mayhood, Todd; Gill, Charles; Mann, Paul; Sher, Xinwei; Ha, Sookhee; Yang, Shu-Wei; Mandal, Mihir; Yang, Christine; Liang, Lianzhu; Tan, Zheng; Tawa, Paul; Hou, Yan; Kuvelkar, Reshma; DeVito, Kristine; Wen, Xiujuan; Xiao, Jing; Batchlett, Michelle; Balibar, Carl J; Liu, Jenny; Xiao, Jianying; Murgolo, Nicholas; Garlisi, Charles G; Sheth, Payal R; Flattery, Amy; Su, Jing; Tan, Christopher; Roemer, Terry

    2016-03-01

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

  2. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci.

    PubMed

    Lee, Sang Ho; Wang, Hao; Labroli, Marc; Koseoglu, Sandra; Zuck, Paul; Mayhood, Todd; Gill, Charles; Mann, Paul; Sher, Xinwei; Ha, Sookhee; Yang, Shu-Wei; Mandal, Mihir; Yang, Christine; Liang, Lianzhu; Tan, Zheng; Tawa, Paul; Hou, Yan; Kuvelkar, Reshma; DeVito, Kristine; Wen, Xiujuan; Xiao, Jing; Batchlett, Michelle; Balibar, Carl J; Liu, Jenny; Xiao, Jianying; Murgolo, Nicholas; Garlisi, Charles G; Sheth, Payal R; Flattery, Amy; Su, Jing; Tan, Christopher; Roemer, Terry

    2016-03-01

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci. PMID:26962156

  3. Effects of norflurazon, an inhibitor of carotenogenesis, on abscisic acid and xanthoxin in the caps of gravistimulated maize roots

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Sun, P. S.

    1986-01-01

    Maize seeds were germinated in the dark in the presence of the carotenoid synthesis inhibitor norflurazon and the levels of abscisic acid, xanthoxin and total carotenoids were measured in the root cap and in the adjacent 1.5 mm segment. In norflurazon-treated roots abscisic acid levels were markedly reduced, but an increase occurred in the levels of xanthoxin, a compound structurally and physiologically similar to abscisic acid. In the cultivar of maize (Zea mays L. cv. Merit) used for this work, brief illumination of the root is required for gravitropic curving. Following illumination both control and norflurazon-treated roots showed normal gravitropic curvature; however, the rate of curvature was delayed in norflurazon-treated roots. Our data from norflurazon-treated roots are consistent with a role for xanthoxin in maize root gravitropism. The increase in xanthoxin in the presence of an inhibitor of carotenoid synthesis suggests that xanthoxin and abscisic acid originate, at least in part, via different metabolic pathways.

  4. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

    PubMed Central

    Forsberg, Kevin J.; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D.

    2015-01-01

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. PMID:26546427

  5. Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

    PubMed

    Zeng, X; Farrenkopf, B; Hohmann, S; Dyda, F; Furey, W; Jordan, F

    1993-03-16

    Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Quantum mechanics/molecular mechanics modeling of fatty acid amide hydrolase reactivation distinguishes substrate from irreversible covalent inhibitors.

    PubMed

    Lodola, Alessio; Capoferri, Luigi; Rivara, Silvia; Tarzia, Giorgio; Piomelli, Daniele; Mulholland, Adrian; Mor, Marco

    2013-03-28

    Carbamate and urea derivatives are important classes of fatty acid amide hydrolase (FAAH) inhibitors that carbamoylate the active-site nucleophile Ser241. In the present work, the reactivation mechanism of carbamoylated FAAH is investigated by means of a quantum mechanics/molecular mechanics (QM/MM) approach. The potential energy surfaces for decarbamoylation of FAAH covalent adducts, derived from the O-aryl carbamate URB597 and from the N-piperazinylurea JNJ1661610, were calculated and compared to that for deacylation of FAAH acylated by the substrate oleamide. Calculations show that a carbamic group bound to Ser241 prevents efficient stabilization of transition states of hydrolysis, leading to large increments in the activation barrier. Moreover, the energy barrier for the piperazine carboxylate was significantly lower than that for the cyclohexyl carbamate derived from URB597. This is consistent with experimental data showing slowly reversible FAAH inhibition for the N-piperazinylurea inhibitor and irreversible inhibition for URB597.

  7. Ellagic acid and polyhydroxylated urolithins are potent catalytic inhibitors of human topoisomerase II: an in vitro study.

    PubMed

    Furlanetto, Valentina; Zagotto, Giuseppe; Pasquale, Riccardo; Moro, Stefano; Gatto, Barbara

    2012-09-12

    Ellagic acid (EA), a natural polyphenol abundant in fruits and common in our diet, is under intense investigation for its chemopreventive activity resulting from multiple effects. EA inhibits topoisomerase II, but the effects on the human enzyme of urolithins, its monolactone metabolites, are not known. Therefore, the action of several synthetic urolithins toward topoisomerases II was evaluated, showing that polyhydroxylated urolithins, EA, and EA-related compounds are potent inhibitors of the α and β isoforms of human topoisomerase II at submicromolar concentrations. Competition tests demonstrate a dose-dependent relationship between ATP and the inhibition of the enzyme. Docking experiments show that the active compounds bind the ATP pocket of the human enzyme, thus supporting the hypothesis that EA and polyhydroxylated urolithins act as ATP-competitive inhibitors of human topoisomerase II.

  8. Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

    PubMed

    Abbassi, Shakeel J; Vishwakarma, Rishi K; Patel, Parth; Kumari, Uma; Khan, Bashir M

    2015-08-01

    Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+).

  9. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    SciTech Connect

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J

    2010-01-01

    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  10. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

    PubMed

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem

    2015-01-01

    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

  11. 2-aminohydroxamic acid derivatives as inhibitors of Bacillus cereus phosphatidylcholine preferred phospholipase C PC-PLC(Bc).

    PubMed

    González-Bulnes, Patricia; González-Roura, Albert; Canals, Daniel; Delgado, Antonio; Casas, Josefina; Llebaria, Amadeu

    2010-12-15

    Phosphatidylcholine preferring phospholipase C (PC-PLC) is an important enzyme that plays a key role in a variety of cellular events and lipid homoeostases. Bacillus cereus phospholipase C (PC-PLC(Bc)) has antigenic similarity with the elusive mammalian PC-PLC, which has not thus far been isolated and purified. Therefore the discovery of inhibitors of PC-PLC(Bc) is of current interest. Here, we describe the synthesis and biological evaluation of a new type of compounds inhibiting PC-PLC(Bc). These compounds have been designed by evolution of previously described 2-aminohydroxamic acid PC-PLC(Bc) inhibitors that block the enzyme by coordination of the zinc active site atoms present in PC-PLC(Bc) [Gonzalez-Roura, A.; Navarro, I.; Delgado, A.; Llebaria, A.; Casas, J. Angew. Chem. Int. Ed.2004, 43, 862]. The new compounds maintain the zinc coordinating groups and possess an extra trimethylammonium function, linked to the hydroxyamide nitrogen by an alkyl chain, which is expected to mimic the trimethylammonium group of the phosphatidylcholine PC-PLC(Bc) substrates. Some of the compounds described inhibit the enzyme with IC(50)'s in the low micromolar range. Unexpectedly, the most potent inhibitors found are those that possess a trimethylammonium group but have chemically blocked the zinc coordinating functionalities. The results obtained suggest that PC-PLC(Bc) inhibition is not due to the interaction of compounds with the phospholipase catalytic zinc atoms, but rather results from the inhibitor cationic group recognition by the PC-PLC(Bc) amino acids involved in choline lipid binding.

  12. Characterization of a novel inhibitor of human DNA polymerases: 3,4,5-tri-O-galloylquinic acid.

    PubMed

    Parker, W B; Nishizawa, M; Fisher, M H; Ye, N; Lee, K H; Cheng, Y C

    1989-11-01

    Various galloyl derivatives of quinic acid were found to be inhibitors of human DNA polymerases. Among them, 3,4,5-tri-O-galloylquinic acid (TGQA) was the most potent inhibitor of DNA polymerase alpha. Under identical conditions, this compound was 60-fold more potent than aphidicolin as an inhibitor of DNA polymerase alpha. The inhibition of DNA polymerase alpha by this compound was not competitive with either the template or any of the deoxynucleoside triphosphates with a Ki of 0.28 microM. Under similar reaction conditions, DNA polymerases beta and gamma were much less sensitive to the effects of these compounds and, in contrast to the effect seen with DNA polymerase alpha, the inhibition of DNA polymerases beta and gamma by TGQA was competitive with respect to the template with Ki values of 44.4 and 7.5 microM respectively. The potency of these compounds against DNA polymerase gamma varied according to the assay conditions used. The inhibition of DNA polymerase gamma by TGQA could be increased substantially by using MnCl2 in place of MgCl2 and by including 50 mM potassium phosphate, pH 7.5, in the assay mixture. DNA polymerase beta was also more sensitive to TGQA when measured with MnCl2. However, potassium phosphate had little, if any, effect on the inhibition by TGQA of either DNA polymerase alpha or beta. DNA polymerase alpha was less sensitive to TGQA when assayed with MnCl2. TGQA was not a potent inhibitor of human KB cell growth in culture, which could be due to its degradation or poor uptake. Nevertheless, this compound could serve as a model for developing antitumor drugs targeted at DNA polymerases.

  13. Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Tsai, Yo-Hsian; Lin, Kuan-Lian; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chen, Chung-Hwan; Chen, Yuhsin; Sie, Min-Hua; Wang, Gwo-Jaw; Lee, Mon-Juan

    2015-07-22

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis. Suppression of ODC by its irreversible inhibitor, α-difluoromethylornithine (DFMO), or by RNA interference through siRNA, enhanced osteogenic gene expression and alkaline phosphatase activity, and accelerated matrix mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs). Besides, adipogenic gene expression and lipid accumulation was attenuated, indicating that the enhanced osteogenesis was accompanied by down-regulation of adipogenesis when ODC was suppressed. A decrease in the intracellular polyamine content of hBMSCs during osteogenic induction was observed, suggesting that the level of endogenous polyamines is regulated during differentiation of hBMSCs. This study elucidates the role of polyamine metabolism in the lineage commitment of stem cells and provides a potential new indication for DFMO as bone-stimulating drug. PMID:26140984

  14. Oral putrescine restores virulence of ornithine decarboxylase-deficient Leishmania donovani in mice

    PubMed Central

    Olenyik, Tamara; Gilroy, Caslin; Ullman, Buddy

    2011-01-01

    Administration of putrescine as a 1% solution in the drinking water ameliorated the profound loss of virulence exhibited by ornithine decarboxylase (ODC) deficient Leishmania donovani in mice. Furthermore, supplying α-difluoromethylornithine, an ODC inhibitor, at 2% in the drinking water reduced but did not eliminate infection with wild type L. donovani in the mouse model. Taken collectively, these findings: 1) demonstrate that oral putrescine can access the phagolysosome of macrophages in which the parasite resides in mice; 2) establish that the loss of virulence due to the Δodc lesion is a consequence of the inability of the mutant parasite to synthesize sufficient polyamines de novo; 3) imply that the L. donovani amastigote cannot access host polyamines in sufficient amounts for survival and growth; 4) and validate ODC as a drug target, although oral administration of DFMO is an unlikely therapeutic paradigm for visceral leishmaniasis. PMID:21182873

  15. Anti-inflammatory effects of 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester, potential inhibitors of neuropeptide bioactivation.

    PubMed

    Bauer, John D; Sunman, Jeffrey A; Foster, Michael S; Thompson, Jeremy R; Ogonowski, Alison A; Cutler, Stephen J; May, Sheldon W; Pollock, Stanley H

    2007-03-01

    Substance P (SP) and calcitonin gene-related peptide (CGRP) are well established mediators of inflammation. Therefore, inhibition of the biosynthesis of these neuropeptides is an attractive potential strategy for pharmacological intervention against a number of inflammatory diseases. The final step in the biosynthesis of SP and CGRP is the conversion of their glycine-extended precursors to the active amidated peptide, and this process is catalyzed by sequential action of the enzymes peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase. We have demonstrated previously that 4-phenyl-3-butenoic acid (PBA) is a PAM inhibitor, and we have also shown that in vivo inhibition of serum PAM by PBA correlates with this compound's ability to inhibit carrageenan-induced edema in the rat. Here we demonstrate the ability of PBA to inhibit all three phases of adjuvant-induced polyarthritis (AIP) in rats; this represents the first time that an amidation inhibitor has been shown to be active in a model of chronic inflammation. We recently introduced 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid (AOPHA) as one of a new series of mechanism-based amidation inhibitors. We now report for the first time that AOPHA and its methyl ester (AOPHA-Me) are active inhibitors of serum PAM in vivo, and we show that AOPHA-Me correspondingly inhibits carrageenan-induced edema in rats in a dose-dependent manner. Neither PBA nor AOPHA-Me exhibits significant cyclooxygenase (COX) inhibition in vitro; thus, the anti-inflammatory activities of PBA and AOPHA-Me are apparently not a consequence of COX inhibition. We discuss possible pharmacological mechanisms that may account for the activities of these new anti-inflammatory compounds.

  16. Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198.

    PubMed

    Lau, Gloria; Labrecque, Jean; Metz, Markus; Vaz, Roy; Fricker, Simon P

    2015-04-24

    The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

  17. Anti-AIDS agents, 1. Isolation and characterization of four new tetragalloylquinic acids as a new class of HIV reverse transcriptase inhibitors from tannic acid.

    PubMed

    Nishizawa, M; Yamagishi, T; Dutschman, G E; Parker, W B; Bodner, A J; Kilkuskie, R E; Cheng, Y C; Lee, K H

    1989-01-01

    Four new tetragalloylquinic acids, 3,5-di-O-galloyl-4-O-digalloylquinic acid, 3,4-di-O-galloyl-5-O-digalloylquinic acid, 3-O-digalloyl-4,5-di-O-galloylquinic acid, and 1,3,4,5-tetra-O-galloylquinic acid, were isolated and characterized from a commercial tannic acid as a new class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitor. Compounds 2, 3, and 4 inhibit HIV RT activity 90, 89, and 84% at 100 microM and 73, 70, and 63% at 30 microM, respectively. Compounds 2-5 also inhibit the HIV growth in cells in the range of 61-70% with low cytotoxicity at 25 microM. The HIV cell growth inhibitory effects of these compounds at 25 microM and 6.25 microM (44-57%) are comparable to their effects against the HIV RT at 30 microM and 10 microM, respectively. The inhibitory effect of 3 against DNA polymerases indicates that the selective antiviral action of 3 is determined by more than its action with HIV RT.

  18. Outcomes in patients with nonerosive reflux disease treated with a proton pump inhibitor and alginic acid ± glycyrrhetinic acid and anthocyanosides

    PubMed Central

    Di Pierro, Francesco; Gatti, Mario; Rapacioli, Giuliana; Ivaldi, Leandro

    2013-01-01

    Background The purpose of this study was to compare the efficacy of alginic acid alone versus alginic acid combined with low doses of pure glycyrrhetinic acid and bilberry anthocyanosides as an addon to conventional proton pump inhibitor therapy in relieving symptoms associated with nonerosive reflux disease. Methods This prospective, randomized, 8-week, open-label trial was conducted at two centers. Sixty-three patients with persistent symptoms of gastroesophageal reflux disease and normal upper gastrointestinal endoscopy were eligible for the study. Patients in group A (n = 31) were treated with pantoprazole and a formula (Mirgeal®) containing alginic acid and low doses of pure glycyrrhetinic acid + standardized Vaccinium myrtillus extract for 4 weeks, then crossed over to the multi-ingredient formula for a further 4 weeks. Patients in group B (n = 32) were treated pantoprazole and alginic acid alone twice daily, then crossed over to alginic acid twice daily for a further 4 weeks. Efficacy was assessed by medical evaluation of a symptom relief score, estimated using a visual analog scale (0–10). Side effects, tolerability, and compliance were also assessed. Results Of the 63 patients enrolled in the study, 58 (29 in group A and 29 in group B) completed the 8-week trial. The baseline characteristics were comparable between the two groups. During the study, significant differences were recorded in symptom scores for both groups. In group A, symptoms of chest pain, heartburn, and abdominal swelling were less serious than in group B. Treatment A was better tolerated, did not induce hypertension, and had fewer side effects than treatment B. No significant differences in compliance were found between the two groups. Conclusion Use of low doses of pure glycyrrhetinic acid + bilberry anthocyanosides, together with alginic acid as addon therapy, substantially improves symptoms in patients with nonerosive reflux disease without increasing side effects or worsening

  19. Potent and Selective α-Ketoheterocycle-Based Inhibitors of the Anandamide and Oleamide Catabolizing Enzyme, Fatty Acid Amide Hydrolase

    PubMed Central

    Romero, F. Anthony; Du, Wu; Hwang, Inkyu; Rayl, Thomas J.; Kimball, F. Scott; Leung, Donmienne; Hoover, Heather S.; Apodaca, Richard L.; Breitenbucher, J. Guy; Cravatt, Benjamin F.; Boger, Dale L.

    2008-01-01

    A study of the structure–activity relationships (SAR) of 2f (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed targeting the 5-position of the oxazole. Examination of a series of substituted benzene derivatives (12–14) revealed that the optimal position for substitution was the meta-position with selected members approaching or exceeding the potency of 2f. Concurrent with these studies, the effect of substitution on the pyridine ring of 2f was also examined. A series of small, non-aromatic C5-substituents was also explored and revealed that the Ki follows a well-defined correlation with the Hammett σp constant (ρ = 3.01, R2 = 0.91) in which electron-withdrawing substituents enhance potency leading to inhibitors with Ki’s as low as 400 pM (20n). Proteomic-wide screening of the inhibitors revealed that most are exquisitely selective for FAAH over all other mammalian proteases reversing the 100-fold preference of 20a (C5 substituent = H) for the enzyme TGH. PMID:17279740

  20. Renoprotective effect of DPP-4 inhibitors against free fatty acid-bound albumin-induced renal proximal tubular cell injury.

    PubMed

    Tanaka, Yuki; Kume, Shinji; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi; Ugi, Satoshi; Sugaya, Takeshi; Uzu, Takashi; Maegawa, Hiroshi

    2016-02-12

    Dipeptidyl peptidase (DPP)-4 inhibitors, a new class of antidiabetic agent, have recently been suggested to exert pleiotropic effects beyond glucose lowering. Renal prognosis in patients with diabetic nephropathy depends on the severity of tubulointerstitial injury induced by massive proteinuria. We thus examined the renoprotective effect of DPP-4 inhibitors on inflammation in cultured mouse proximal tubular cells stimulated with free fatty acid (FFA)-bound albumin. Linagliptin and higher concentrations of sitagliptin, vildagliptin, and alogliptin all inhibited FFA-bound albumin-induced increases in mRNA expression of MCP-1 in cultured mouse proximal tubular cells. Furthermore, linagliptin significantly inhibited tubulointerstitial injury induced by peritoneal injection of FFA-bound albumin, such as inflammation, fibrosis, and apoptosis, in mice without altering systemic characteristics including body weight, fasting blood glucose, and food intake. These results indicate that DPP-4 inhibitors pleiotropically exert a direct renoprotective effect, and may serve as an additional therapeutic strategy to protect proximal tubular cells against proteinuria in patients with diabetic nephropathy. PMID:26802469

  1. Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases with antiviral activity against human respiratory syncytial virus.

    PubMed

    Cui, Rui; Wang, Yizhuo; Wang, Liu; Li, Guiming; Lan, Ke; Altmeyer, Ralf; Zou, Gang

    2016-08-01

    Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants and young children worldwide, yet no vaccine or effective antiviral treatment is available. To search for new anti-RSV agents, we developed a cell-based assay that measures inhibition of RSV-induced cytopathic effect (CPE) and identified cyclopiazonic acid (CPA), an intracellular calcium ATPase inhibitor as a RSV inhibitor (EC50 values 4.13 μM) by screening of natural product library. CPA inhibited the replication of RSV strains belonging to both A and B subgroups and human parainfluenza virus type 3, but not Enterovirus 71. Mechanism of action study by time-of-addition assay and minigenome assay revealed that CPA acts at the step of virus genome replication and/or transcription. Moreover, two other calcium ATPase inhibitors (Thapsigargin and BHQ) and calcium ionophores (A23187 and ionomycin), but not calcium channel blockers (nifedipine, nimodipine, and tetrandrine), also had similar effect. These results indicate that an increase in intracellular calcium concentration is detrimental to RSV replication. Thus, our findings provide a new strategy for anti-RSV therapy via increasing intracellular calcium concentration. PMID:27210812

  2. Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    PubMed Central

    Zhang, Laitao; Li, Yi-Fang; Yuan, Sheng; Zhang, Shijie; Zheng, Huanhuan; Liu, Jie; Sun, Pinghua; Gu, Yijun; Kurihara, Hiroshi; He, Rong-Rong; Chen, Heru

    2016-01-01

    Bioactivity focus on α-cyano-4-hydroxycinnamic acid (CHCA) scaffold results in a small library of novel multifunctional aldose reductase (ALR2) inhibitors. All the entities displayed good to excellent inhibition with IC50 72–405 nM. (R,E)-N-(3-(2-acetamido-3-(benzyloxy)propanamido)propyl)-2-cyano-3-(4-hydroxy phenyl)acrylamide (5f) was confirmed as the most active inhibitor (IC50 72.7 ± 1.6 nM), and the best antioxidant. 5f bound to ALR2 with new mode without affecting the aldehyde reductase (ALR1) activity, implicating high selectivity to ALR2. 5f was demonstrated as both an effective ALR2 inhibitor (ARI) and antioxidant in a chick embryo model of hyperglycemia. It attenuated hyperglycemia-induced incidence of neural tube defects (NTD) and death rate, and significantly improved the body weight and morphology of the embryos. 5f restored the expression of paired box type 3 transcription factor (Pax3), and reduced the hyperglycemia-induced increase of ALR2 activity, sorbitol accumulation, and the generation of ROS and MDA to normal levels. All the evidences support that 5f may be a potential agent to treat diabetic complications. PMID:27109517

  3. The macamide N-3-methoxybenzyl-linoleamide is a time-dependent fatty acid amide hydrolase (FAAH) inhibitor.

    PubMed

    Almukadi, Haifa; Wu, Hui; Böhlke, Mark; Kelley, Charles J; Maher, Timothy J; Pino-Figueroa, Alejandro

    2013-10-01

    The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide, was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters. PMID:23853040

  4. Potentiation of antinociceptive effect of NSAIDs by a specific lipooxygenase inhibitor, acetyl 11-keto-beta boswellic acid.

    PubMed

    Bishnoi, Mahendra; Patil, Chandrashekar S; Kumar, Anil; Kulkarni, Shrinivas K

    2006-02-01

    The present study was aimed to assess the combined effects of cyclooxygenase and 5-lipoxygenase (COX/5-LOX) inhibitors in different animal models of nociception. Naproxen, nimesulide and rofecoxib are well-established antinociceptive agents acting via COX inhibition. AKBA (acetyl-keto-beta-boswellic acid) is a 5-LOX inhibitor. AKBA (50-200 mg/kg) produced a dose dependent and significant antinociceptive effect in different animal models of nociception. Based on the earlier reports from our laboratory, sub effective doses of all the three COX Inhibitors were selected and they were administered (naproxen, 5 mg/kg; nimesulide, 1 mg/kg; and rofecoxib, 1 mg/kg) with AKBA (100 mg/kg). This produced a more significant antinociceptive effect as compared to per se effect observed in all the three models of nociception. However, the effect of combination of nimesulide with AKBA was more pronounced as compared to naproxen and rofecoxib and their combination with AKBA. The present finding provided an evidence for the potentiation of antinociceptive effect of NSAIDs with AKBA. Such a combination may help to reduce the therapeutic doses of conventional NSAIDs and also reduce side effects (gastric, cardiac and renal) that are popularly associated with the NSAIDs.

  5. Identification of indole-3-carboxylic acids as non-ATP-competitive Polo-like kinase 1 (Plk1) inhibitors.

    PubMed

    Liu, Meng; Huang, Jie; Chen, Dong-Xing; Jiang, Cheng

    2015-02-01

    A series of indole-3-carboxylic acids were designed as novel small molecular non-ATP-competitive Plk1 inhibitors. The designed compounds were synthesized and evaluated. Most of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 4f and 4g showed Plk1 inhibitory activity with IC50 values of 0.41 and 0.13μM, which were about 5 and 17 times more potent compared to thymoquinone, respectively. Compound 4g also showed inhibitory activity to HeLa and MCF-7 cell lines with IC50 values of 0.72 and 1.15μM, which was almost 3 and 4 times more potent than thymoquinone. Study of mechanism of action suggested that 4g was an ATP-independent and substrate-dependent Plk1 inhibitor. Moreover, 4g showed excellent Plk1 inhibitory selectivity against Plk2 and Plk3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. This study may provide a support for further optimization of non-ATP-competitive Plk1 inhibitors.

  6. Potential inhibitors of L-asparagine biosynthesis. 5. Electrophilic amide analogues of (S)-2,3-diaminopropionic acid.

    PubMed

    Mokotoff, M; Logue, L W

    1981-05-01

    Three electrophilic amide analogues of (S)-2,3-diaminopropionic acid (1, DAP) have been prepared as potential inhibitors of L-asparagine synthetase (ASase, from Novikoff hepatoma, EC 6.3.5.4). DAP was selectively blocked by the carbobenzoxy (Cbz) group to give 3-N-Cbz-DAP (2a). Esterification of 2a with isobutylene afforded tert-butyl 3-N-carbobenzoxy-(S)-2,3-diaminopropionate (3a), which was then blocked at the 2 position with the tert-butoxycarbonyl (Boc) group to give tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-[(carbobenzoxy)amino]propionate (4). Selective cleavage of the Cbz group by H2/Pd gave the key intermediate tert-butyl 2-N-(tert-butoxycarbonyl)-(S)-2,3-diaminopropionate (5), which was acylated, via the N-hydroxysuccinimide esters, with bromoacetic acid, dichloroacetic acid, and fumaric acid monoethyl ester to give tert-butyl 2-[(S)-(tert-butoxycarbonyl)-amino]-3-(2-bromoacetamido)propionate (6a), tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-(2,2-dichloroacetamido)propionate (6b), and tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-(ethoxycarbonyl)acrylamido]-propionate (6c), respectively. Deblocking of 6a-c gave the corresponding amino acids (S)-2-amino-3-(2-bromoacetamido)propionic acid hydrobromide (7a), (S)-2-amino-3-(2,2-dichloroacetamido)propionic acid (7b), and ethyl N-[(S)-2-amino-2-carboxyethyl]fumarate (7c). By a slightly different procedure, 5 was converted in two steps to (S)-2-amino-3-acetamidopropionic acid hydrobromide (7d). The inhibition of ASase by 7a-c at 1 mM was 93, 19, and 37%, respectively, while 7d was without inhibition at 2 mM. Compounds 7a-c failed to increase the life span of mice infected with B16 melanoma.

  7. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety.

    PubMed

    Nakayama, Kiyoshi; Kuru, Noriko; Ohtsuka, Masami; Yokomizo, Yoshihiro; Sakamoto, Atsunobu; Kawato, Haruko; Yoshida, Ken-ichi; Ohta, Toshiharu; Hoshino, Kazuki; Akimoto, Katsuya; Itoh, Junko; Ishida, Hiroko; Cho, Aesop; Palme, Monica H; Zhang, Jason Z; Lee, Ving J; Watkins, William J

    2004-05-17

    Exchange of the ethylene tether in a series of pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors to an amide bond stabilized the olefin of the acrylic acid moiety, preventing facile photoisomerization to the Z-isomer. Furthermore, the activity was drastically improved in the amide tether variants, providing extremely potent acrylic acid and vinyl tetrazole analogues.

  8. The natural diyne-furan fatty acid EV-086 is an inhibitor of fungal delta-9 fatty acid desaturation with efficacy in a model of skin dermatophytosis.

    PubMed

    Knechtle, Philipp; Diefenbacher, Melanie; Greve, Katrine B V; Brianza, Federico; Folly, Christophe; Heider, Harald; Lone, Museer A; Long, Lisa; Meyer, Jean-Philippe; Roussel, Patrick; Ghannoum, Mahmoud A; Schneiter, Roger; Sorensen, Alexandra S

    2014-01-01

    Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida, Aspergillus, and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae, depending on the delta-9 fatty acid desaturase OLE1. Delta-9 unsaturated fatty acids-but not saturated fatty acids-antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086-phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses.

  9. Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors.

    PubMed

    De Lucia, Daniela; Lucio, Oscar Méndez; Musio, Biagia; Bender, Andreas; Listing, Monika; Dennhardt, Sophie; Koeberle, Andreas; Garscha, Ulrike; Rizzo, Roberta; Manfredini, Stefano; Werz, Oliver; Ley, Steven V

    2015-08-28

    In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 μm in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization.

  10. Effects of an inhibitor of poly(ADP-ribose) polymerase, desmethylselegiline, trientine, and lipoic acid in transgenic ALS mice.

    PubMed

    Andreassen, O A; Dedeoglu, A; Friedlich, A; Ferrante, K L; Hughes, D; Szabo, C; Beal, M F

    2001-04-01

    The development of transgenic mouse models of amyotrophic lateral sclerosis (ALS) allows the testing of neuroprotective agents. We evaluated the effects of five agents in transgenic mice with the G93A Cu,Zn superoxide dismutase mutation. A novel inhibitor of poly(ADP-ribose) polymerase showed no effects on survival. Desmethylselegiline and CGP3466 are agents that exert antiapoptotic effects in vitro by preventing nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase. They had no significant effects on survival in the G93A mice. Trientine, a copper chelator, produced a modest significant increase in survival. Similarly administration of lipoic acid in the diet produced a significant improvement in survival. These results therefore provide evidence for potential therapeutic effects of copper chelators and lipoic acid in the treatment of ALS.

  11. The pharmacology and therapeutic potential of small molecule inhibitors of acid-sensing ion channels in stroke intervention

    PubMed Central

    Leng, Tian-dong; Xiong, Zhi-gang

    2013-01-01

    In the nervous system, a decrease in extracellular pH is a common feature of various physiological and pathological processes, including synaptic transmission, cerebral ischemia, epilepsy, brain trauma, and tissue inflammation. Acid-sensing ion channels (ASICs) are proton-gated cation channels that are distributed throughout the central and peripheral nervous systems. Following the recent identification of ASICs as critical acid-sensing extracellular proton receptors, growing evidence has suggested that the activation of ASICs plays important roles in physiological processes such as nociception, mechanosensation, synaptic plasticity, learning and memory. However, the over-activation of ASICs is also linked to adverse outcomes for certain pathological processes, such as brain ischemia and multiple sclerosis. Based on the well-demonstrated role of ASIC1a activation in acidosis-mediated brain injury, small molecule inhibitors of ASIC1a may represent novel therapeutic agents for the treatment of neurological disorders, such as stroke. PMID:22820909

  12. Polymerized liposomes containing C-glycosides of sialic acid: Potent inhibitors of influenza virus in vitro infectivity

    SciTech Connect

    Spevak, W.; Bednarski, M.D.; Nagy, J.O.; Charych, D.H ); Schaefer, M.E.; Gilbert, J.H. )

    1993-02-10

    The surface lectin of the influenza virus, hemagglutinin, binds to terminal [alpha]-glycosides of N-acetylneuraminic acid (NeuAc) on cell-surface glycoproteins and glycolipids. Viral binding to cells expression terminal NeuAc residues can be inhibited by [alpha]-O-glycosides of NeuAc (O-sialosides). Recently, dramatic enhancements in the inhibition of viral adhesion to erythrocytes have been achieved using synthetic polyvalent sialosides. In this communication, the authors report that polymerized liposomes containing [alpha]-C-glycosides of sialic acid are potent inhibitors of influenza virus in vitro infectivity. Their results also indicate that the capacity to inhibit hemagglutination does not necessarily reflect the capacity to inhibit in vitro infectivity. 14 refs., 1 tab.

  13. Assaying Ornithine and Arginine Decarboxylases in Some Plant Species 1

    PubMed Central

    Birecka, Helena; Bitonti, Alan J.; McCann, Peter P.

    1985-01-01

    A release of 14CO2 not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-14C]- or [U-14C]ornithine. The total 14CO2 produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH4)2SO4 did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant 14CO2 release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris·HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard 14CO2-based method by assessing labeled Put and/or Agm—formed in the presence of added aminoguanidine and/or ornithine when needed—is recommended especially when crude or semicrude plant extracts are assayed. When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids. PMID:16664441

  14. Design, synthesis and SAR studies of GABA uptake inhibitors derived from 2-substituted pyrrolidine-2-yl-acetic acids.

    PubMed

    Steffan, Tobias; Renukappa-Gutke, Thejavathi; Höfner, Georg; Wanner, Klaus T

    2015-03-15

    In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The 2-position in the side chain of pyrrolidine-2-yl-acetic acid derivatives was substituted with alkyl, hydroxy and amino groups to modulate the activity and selectivity to mGAT1 and mGAT4 proteins. SAR studies of the compounds performed for the four mouse GABA transporter proteins (mGAT1-mGAT4) implied significant potencies and subtype selectivities for 2-hydroxy-2-pyrrolidine-2-yl-acetic acid derivatives. The racemate rac-(u)-13c exhibited the highest potency (pIC50 5.67) at and selectivity for mGAT1 in GABA uptake assays. In fact, the potency of rac-(u)-13c at hGAT-1 (pIC50 6.14) was even higher than its potency at mGAT1. These uptake results for rac-(u)-13c are in line with the binding affinities to the aforesaid proteins mGAT1 (pKi 6.99) and hGAT-1 (pKi 7.18) determined by MS Binding Assay based on NO711 as marker quantified by LC-ESI-MS-MS analysis. Interestingly, the 2-hydroxy-2-pyrrolidine-2-yl-acetic acid rac-(u)-13d containing 2-{[tris(4-methoxyphenyl)]methoxy} ethyl group at the nitrogen atom of the pyrrolidine ring showed high potency at mGAT4 and a comparatively better selectivity for this protein (>15 against mGAT3) than the well known mGAT4 uptake inhibitor (S)-SNAP-5114.

  15. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    PubMed

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  16. The hydratase activity of malonate semialdehyde decarboxylase: mechanistic and evolutionary implications.

    PubMed

    Poelarends, Gerrit J; Serrano, Hector; Johnson, William H; Hoffman, David W; Whitman, Christian P

    2004-12-01

    Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic beta-alpha-beta-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a kcat/Km value of 6.0 x 102 M-1 s-1. Pro-1 and Arg-75 are critical for both activities, and the pKa of Pro-1 was determined to be approximately 9.2 by a direct 15N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pKa value of Pro-1 are shared with trans-3-chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water.

  17. Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acids as potent xanthine oxidase inhibitors.

    PubMed

    Guan, Qi; Cheng, Zengjin; Ma, Xiaoxue; Wang, Lijie; Feng, Dongjie; Cui, Yuanhang; Bao, Kai; Wu, Lan; Zhang, Weige

    2014-10-01

    A series of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid derivatives (8a-f, 9a-m) were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Structure-activity relationship analyses have also been presented. Most of the target compounds exhibited potency levels in the nanomolar range. Compound 9e emerged as the most potent xanthine oxidase inhibitor (IC50 = 5.5 nM) in comparison to febuxostat (IC50 = 18.6 nM). Steady-state kinetics measurements with the bovine milk enzyme indicated a mixed type inhibition with Ki and Ki' values of 0.9 and 2.3 nM, respectively. A molecular modeling study on compounds 9e was performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors related with 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid scaffold.

  18. Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease.

    PubMed

    Civallero, G; De Mari, J; Bittar, C; Burin, M; Giugliani, R

    2014-04-10

    Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p<0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann-Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.

  19. Effects of tumour necrosis factor-alpha synthesis inhibitors on rat trinitrobenzene sulphonic acid-induced chronic colitis.

    PubMed

    Bobin-Dubigeon, C; Collin, X; Grimaud, N; Robert, J M; Le Baut, G; Petit, J Y

    2001-11-01

    The fact that tumour necrosis factor-alpha (TNF-alpha) is clearly involved in the pathogenesis of intestinal bowel disease, especially Crohn's disease, suggests that TNF-alpha synthesis inhibitors could be beneficial for treatment. The present study assessed the effect of chronic oral gavage of two in vitro TNF-alpha synthesis inhibitors, JM 34 maleate or [N-(4,6-dimethylpyridin-2-yl)-furane-2-carboxamide)] maleate and XC 21 or (N-betapicolyl-tetrafluorophtalimide), on colonic inflammation in trinitrobenzene sulphonic acid-induced colitis in rats. Rats received JM 34 maleate (100 mg/kg) and XC 21 (50 mg/kg) 1 h before colitis induction and then daily for 8 days by oral gavage. The colon was removed on day 8 and processed for clinical score, myeloperoxidase activity, and soluble TNF-alpha release. Treatment with XC 21, as well as dexamethasone and sulphasalazine, reduced colonic damage and decreased (except with dexamethasone) the incidence of diarrhoea. JM 34 maleate failed to improve the clinical signs of chronic colitis. After trinitrobenzene sulphonic acid-induced colitis, myeloperoxidase activity and TNF-alpha colonic mucosal production were substantially increased compared to the control (saline instillation). Both of these inflammatory indicators were then significantly decreased (P< or =0.05) after the four chronic treatments (JM 34 maleate, XC 21, sulphasalazine, and dexamethasone). XC 21 appeared to be as efficient as sulphasalazine in improving colonic inflammation. PMID:11716848

  20. Complexes of Imidazole with Poly(ethylene glycol) as a Corrosion Inhibitor for Carbon Steel in Sulphuric Acid

    NASA Astrophysics Data System (ADS)

    Salimi, Saeed; Nasr-Esfahani, Mojtaba; Umoren, Saviour A.; Saebnoori, Ehsan

    2015-12-01

    The inhibiting action of polyethylene glycol and imidazole (PEG/IMZ)) complexes prepared by a simple deprotonation procedure on carbon steel corrosion in 0.5 mol/L sulphuric acid was evaluated using the weight loss, potentiodynamic polarization, and electrochemical impedance spectroscopy techniques complemented by surface analysis using scanning electron microscopy. The inhibiting effect of the PEG/IMZ complexes on carbon steel corrosion was compared with the non-complex forms. Results obtained show that PEG/IMZ complex is a very effective corrosion inhibitor of carbon steel in the acid environment. The inhibition efficiency increased with the increase in the temperature and also with increasing percentage of imidazole in the complex. Corrosion inhibition occurs by virtue of adsorption of PEG/IMZ complexes on the steel surface which was found to follow the Temkin adsorption isotherm model. The PEG/IMZ complexes function as a mixed-type inhibitor. Results from all the methods employed are in a reasonably good agreement.

  1. Effects of COX inhibitors on neurodegeneration and survival in mice exposed to the marine neurotoxin domoic acid.

    PubMed

    Ryan, James C; Cross, Cheryl A; Van Dolah, Frances M

    2011-01-01

    The marine neurotoxin domoic acid (DA) is a rigid analogue of the neurotransmitter glutamate and a potent agonist of kainate subtype glutamate receptors. Persistent activation of these receptor subtypes results in rapid excitotoxicity, calcium-dependent cell death, and neuronal degeneration in regions of the brain where glutamatergic pathways are concentrated. Previous work has shown that DA promotes the expression of inflammatory genes in the brain, such as cyclooxygenase 2 (COX2). To investigate the impact of inflammation on the development of neurodegeneration, and ultimately survival following DA administration, we used selective (L745337, Merck) and non-selective (acetylsalicylic acid (ASA)) COX inhibitors in DA exposed mice. Adult male ICR mice were given a regime of either ASA or L23547 both before and after a single LD50 dose of DA. Mice were observed immediately after toxin introduction and then sacrificed at 2 days post exposure. Our lower dose of L23547 increased survival and was most effective at decreasing neuronal degeneration in the CA1 and CA3 regions of the hippocampus, areas especially sensitive to DA excitotoxicity. This study shows that COX2 plays a role in DA induced neurodegeneration and death, and that inhibitors may be of value for treatment in human and wildlife DA exposure. PMID:20934488

  2. Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3.

    PubMed

    Anangi, Raveendra; Chen, Chih-Cheng; Lin, Yi-Wen; Cheng, Yuan-Ren; Cheng, Chun-Ho; Chen, Yi-Chun; Chu, Yuan-Ping; Chuang, Woei-Jer

    2010-12-01

    Acid sensing ion channels (ASICs) are family of proteins predominantly present in the central and peripheral nervous system. They are known to play important roles in the pathophysiology of pain and ischemic stroke. APETx2 is a potent and selective inhibitor of ASIC3-containing channels and was isolated from sea anemone Anthopleura elegantissima. To facilitate the study on the molecular determinants of ASIC3-ligand interactions, we expressed recombinant APETx2 in the Pichia pastoris (P. pastoris) expression system and purified it to homogeneity. Recombinant APETx2 produced in P. pastoris inhibited the acid-evoked ASIC3 current with the IC(50) value of 37.3 nM. The potency of recombinant toxin is similar to that of native APETx2. The sequential assignment and structure analysis of APETx2 were obtained by 2D and 3D (15)N-edited NMR spectra. Our NMR data suggests that APETx2 produced in P. pastoris retained its native fold. The results presented here provide the first direct evidence that highly disulfide bonded peptide inhibitor of ASIC3, APETx2, can be expressed in P. pastoris with correct fold and high yield. We also showed that the R17A mutant exhibited a decrease in activity, suggesting the feasibility of the use of this expression system to study the interactions between APETx2 and ASIC3. These evidences may serve as the basis for understanding the selectivity and activity of APETx2. PMID:20813121

  3. Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse

    PubMed Central

    Justinova, Zuzana; Panlilio, Leigh V; Moreno-Sanz, Guillermo; Redhi, Godfrey H; Auber, Alessia; Secci, Maria E; Mascia, Paola; Bandiera, Tiziano; Armirotti, Andrea; Bertorelli, Rosalia; Chefer, Svetlana I; Barnes, Chanel; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R

    2015-01-01

    Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose–response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell—consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement. PMID:25754762

  4. A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6.

    PubMed

    Peng, Xue; Masai, Eiji; Kasai, Daisuke; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2005-09-01

    A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.

  5. Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors.

    PubMed

    Taddei, Maurizio; Cini, Elena; Giannotti, Luca; Giannini, Giuseppe; Battistuzzi, Gianfranco; Vignola, Davide; Vesci, Loredana; Cabri, Walter

    2014-01-01

    A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC50 values between 2 and 10nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC50=0.5μM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far. PMID:24345446

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

    PubMed

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

    2011-06-14

    Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases. PMID:26596454

  7. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    PubMed Central

    Sadowski, Martin C.; Pouwer, Rebecca H.; Gunter, Jennifer H.; Lubik, Amy A.; Quinn, Ronald J.; Nelson, Colleen C.

    2014-01-01

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

  8. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.

    PubMed

    Sadowski, Martin C; Pouwer, Rebecca H; Gunter, Jennifer H; Lubik, Amy A; Quinn, Ronald J; Nelson, Colleen C

    2014-10-15

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

  9. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  10. Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

    PubMed Central

    Rojas, Laura J.; Taracila, Magdalena A.; Papp-Wallace, Krisztina M.; Bethel, Christopher R.; Caselli, Emilia; Romagnoli, Chiara; Winkler, Marisa L.; Spellberg, Brad; Prati, Fabio

    2016-01-01

    Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of β-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 β-lactamases, nine possessed 50% inhibitory concentrations (IC50s) of ≤600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k2/K) of 1.2 ± 0.2 × 104 M−1 s−1 for KPC-2 and 4.7 ± 0.6 × 103 M−1 s−1 for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or blaKPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A β-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter. PMID:26729496

  11. Effect of inducers and inhibitors of glucuronidation on the biliary excretion and choleretic action of valproic acid in the rat.

    PubMed

    Watkins, J B; Klaassen, C D

    1982-02-01

    Valproic acid (VPA) induces an immediate choleresis in the rat which may be attributable to the osmotic properties of VPA-glucuronic acid conjugates in bile. The influence of inducers and inhibitors of glucuronidation of VPA on the biliary excretion and choleretic effect of VPA was studied. Hepatic UDP-glucuronyltransferase activity toward VPA was determined in vitro. Pretreatment with phenobarbital (75 mg/kg/day for 4 days) enhanced VPA glucuronidation; borneol (750 mg/kg) decreased VPA conjugation; 3-methylcholanthrene (20 mg/kg/day for 4 days) and galactosamine (600 mg/kg) had no effect on glucuronidation of VPA in vitro. Hepatic UDP-glucuronic acid content was decreased by borneol and galactosamine administration and was enhanced by phenobarbital and 3-methylcholanthrene pretreatment. The enzyme inducers increased the plasma disappearance of VPA in vivo but did not augment its biliary excretion or choleretic effect. Borneol and galactosamine, which inhibited the conjugation and plasma disappearance of VPA, decreased its biliary excretion and inhibited the VPA-induced increase in bile flow. Thus, the bile flow rate after VPA administration is closely related to the excretion of VPA-glucuronic acid. These data support the conclusion that the choleretic effect of VPA is due to the osmotic activity of VPA conjugates in bile.

  12. Ursodeoxycholic acid choleresis: Relationship to biliary HCO sup minus sub 3 and effects of Na sup + -H sup + exchange inhibitors

    SciTech Connect

    Renner, E.L.; Lake, J.R.; Cragoe, E.J. Jr.; van Dyke, R.W.; Scharschmidt, B.F. Merck Sharp and Dohme Research Laboratories, West Point, PA )

    1988-02-01

    The authors have recently shown that substitution of Li{sup +} for perfusate Na{sup +} eliminates the HCO{sub 3}{sup {minus}}-rich choleresis produced by ursodeoxycholic acid (UDCA) in isolated perfused rat liver and that the increase in bile flow produced by both UDCA and taurocholic acid is partially inhibited by 1 mM amiloride. Although these findings are consistent with a role for Na{sup +}-H{sup +} exchange in the choleresis produced by these bile acids, both Li{sup +} substitution and amiloride affect other cellular processes, including Na{sup +}-K{sup +}-ATPase activity. They have now further explored both the relationship between UDCA-stimulated bile flow and biliary HCO{sub 3}{sup {minus}} secretion and the possible role of Na{sup +}-H{sup +} exchange in this process by comparing the effects of amiloride with two of its more potent and presumably more specific analogues, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA). None of the inhibitors significantly altered biliary UDCA output or the relationship between UDCA-induced bile flow and either biliary (HCO{sub 3}{sup {minus}}) or biliary HCO{sub 3}{sub {minus}} output. Effects of these inhibitors did not appear attributable either to nonspecific toxicity, as reflected by hepatic release of lactate dehydrogenase or K{sup +}, or to inhibition of hepatic Na{sup +}-K{sup +}-ATPase, measured as Na{sup +}-dependent uptake of {sup 86}Rb. These findings indicate that UDCA-induced but not basal bile formation is closely coupled to biliary HCO{sub 3}{sup {minus}} concentration and output, and they provide additional evidence that UDCA choleresis requires an intact Na{sup +}-H{sup +} exchange mechanism.

  13. Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

    PubMed Central

    Singh, Jaspreet; Khan, Mushfiquddin; Pujol, Aurora; Baarine, Mauhamad; Singh, Inderjit

    2013-01-01

    In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD. PMID:23923017

  14. Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii.

    PubMed

    Peng, Z Y; Mansour, J M; Araujo, F; Ju, J Y; McKenna, C E; Mansour, T E

    1995-01-01

    Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.

  15. Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors.

    PubMed

    Zhao, Hua; Holmes, Shaletha S; Baker, Gary A; Challa, Suresh; Bose, Himangshu S; Song, Zhiyan

    2012-10-01

    Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 μg mL(-1)) was one-third that of wide-type betulinic acid (60 μg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.

  16. Synthesis and Structure-activity Relationship Studies of O-Biphenyl-3-yl Carbamates as Peripherally Restricted Fatty Acid Amide Hydrolase Inhibitors

    PubMed Central

    Moreno-Sanz, Guillermo; Duranti, Andrea; Melzig, Laurin; Fiorelli, Claudio; Ruda, Gian Filippo; Colombano, Giampiero; Mestichelli, Paola; Sanchini, Silvano; Tontini, Andrea; Mor, Marco; Bandiera, Tiziano; Scarpelli, Rita; Tarzia, Giorgio; Piomelli, Daniele

    2014-01-01

    The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3’-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3’-carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date. PMID:23822179

  17. Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in Q(B)-site of Chlamydomonas reinhardtii.

    PubMed

    Chen, Shiguo; Xu, Xiaoming; Dai, Xinbin; Yang, Chunlong; Qiang, Sheng

    2007-04-01

    Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q(A) to Q(B) at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q(B)-niche. The results of competitive replacement with [(14)C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q(B)-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.

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

    PubMed

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

    2011-01-01

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

  19. Identification of geranic acid, a tyrosinase inhibitor in lemongrass (Cymbopogon citratus).

    PubMed

    Masuda, Toshiya; Odaka, Yuka; Ogawa, Natsuko; Nakamoto, Katsuo; Kuninaga, Hideki

    2008-01-23

    Lemongrass is a popular Asian herb having a lemon-like flavor. Very recently, potent tyrosinase inhibitory activity has been found in lemongrass in addition to various biological activities reported in the literature. The aim of the present study is to identify the active compounds in the lemongrass. An assay-guided purification revealed that one of the active substances was geranic acid. Geranic acid has two stereoisomers, which are responsible for the trans and cis geometry on the conjugated double bond. Both isomers are present in the active ethyl acetate-soluble extract of the lemongrass, and their IC50 values were calculated to be 0.14 and 2.3 mM, respectively. The structure requirement of geranic acid for the potent tyrosinase inhibitory activity was investigated using geranic acid-related compounds.

  20. Effect of methionine deprivation on S-adenosylmethionine decarboxylase of tumour cells.

    PubMed

    Tisdale, M J

    1981-07-17

    Transference of Walker carcinoma and TLX5 lymphoma from normal L-methionine-containing medium to medium containing limiting amounts of L-methionine, or L-homocysteine only, caused a 2-fold increase of S-adenosylmethionine decarboxylase activity. Kinetic analysis showed an increase in the V value of the enzyme from 22 to 53 pmol/min per mg protein in media containing only 0.1 mM L-homocysteine, without any alteration in the Km value (0.1 mM). The increase in enzyme activity does not result from (a) a reduction of the intracellular level of S-adenosylmethionine, since cycloleucine, an inhibitor of methionine adenosyltransferase, had no effect on enzyme activity; (b) an increase in intracellular adenosine 3',5' monophosphate (cyclic AMP), since high extracellular concentrations of N6-monobutyryl cyclic AMP had no effect on enzyme activity; (c) an alteration of polyamine levels, since addition of micromolar concentrations of exogenous putrescine, spermidine and spermine did not prevent the induction of S-adenosylmethionine decarboxylase activity in methionine-free media containing 0.1 mM L-homocysteine. The increased enzyme activity appears to be mainly due to enhanced stabilization, since the half-life was increased from 2.45 to 5.0 h in media containing only 0.1 mM L-homocysteine. Induction of enzyme activity is specific to the removal of L-methionine, since no increase occurred in the absence of L-serine or L-glycine, or both, or by reduction of the serum concentrations in the medium.

  1. Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

    PubMed Central

    Hao, Pan; Alaraj, Intisar Q. M.; Dulayymi, Juma’a R. Al; Baird, Mark S.; Liu, Jing; Liu, Qun

    2016-01-01

    Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC50 value of 36.2 μM, compared with EC50 values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis. PMID:27180571

  2. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    SciTech Connect

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for <