An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids

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Frossard, Mariana Lins; Seabra, Sergio Henrique; Matta, Renato Augusto da; Souza, Wanderley de; Garcia de Mello, Fernando; Motta, Maria Cristina Machado

2006-01-01

Summary: Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism

Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

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Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

2006-01-01

The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm

Effects of bis(guanylhydrazones) on the activity and expression of ornithine decarboxylase.

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Nikula, P; Alhonen-Hongisto, L; Jänne, J

1985-01-01

Derivatives of glyoxal bis(guanylhydrazone) (GBG), such as methylglyoxal bis(guanylhydrazone) and ethylglyoxal bis(guanylhydrazone), are potent inhibitors of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the key enzyme required for the synthesis of spermidine and spermine. These compounds, but not the parent compound, induce a massive accumulation of putrescine, partly by blocking the conversion of putrescine into spermidine, but also by strikingly stimulating ornithine decarboxylase (ODC; EC 4.1.1.17) activity. The mechanism of the stimulation of ODC activity and enhanced accumulation of the enzyme protein apparently involved a distinct stabilization of the enzyme against intracellular degradation. However, although the parent compound GBG also stabilized ODC, it powerfully inhibited the enzyme activity and the accumulation of immunoreactive protein in cultured L1210 leukaemia cells. Kinetic considerations indicated that, in addition to the stabilization, all three compounds, GBG in particular, inhibited the expression of ODC. It is unlikely that the decreased rate of synthesis of ODC was attributable to almost unaltered amounts of mRNA in drug-treated cells, thus supporting the view that especially GBG apparently depressed the expression of ODC at some post-transcriptional level. Images PMID:4062886

N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells.

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Medina-Enríquez, Miriam Marlene; Alcántara-Farfán, Verónica; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José Guadalupe; Rodríguez-Páez, Lorena; Vargas-Ramírez, Alba Laura

2015-06-01

Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.

Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

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Rosen, C.F.; Gajic, D.; Drucker, D.J.

1990-01-01

UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation

Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

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Rosen, C.F.; Gajic, D.; Drucker, D.J. (Women' s College Hospital, Toronto, Ontario (Canada))

1990-05-01

UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.

[Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis].

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Logvinovich, O S; Aksenova, G E

2013-01-01

Ornithine decarboxylase (ODC, EC 4.1.1.17.) is a short-lived and dynamically regulated enzyme of polyamines biosynthesis. Regulation of functional, metabolic and proliferative state of organs and tissues involves the modifications of the ODC enzymatic activity. The organ-specific changes in ODC activity were revealed in organs and tissues (liver, spleen, bone marrow, kidney, and intestinal mucosa) of hibernating mammals - squirrels Spermophilus undulates - during the hibernating season. At that, a positive correlation was detected between the decline and recovery of the specialized functions of organs and tissues and the respective modifications of ODC activity during hibernation bouts. Investigation of changes in ODC activity in organs and tissues of non-hibernating mammals under artificial hypobiosis showed that in Wistar rats immediately after exposure to hypothermia-hypoxia-hypercapnia (hypobiosis) the level of ODC activity was low in thymus, spleen, small intestine mucosa, neocortex, and liver. The most marked reduction in enzyme activity was observed in actively proliferating tissues: thymus, spleen, small intestine mucosa. In bone marrow of squirrels, while in a state of torpor, as well as in thymus of rats after exposure to hypothermia-hypoxia-hypercapnia, changes in the ODC activity correlated with changes in the rate of cell proliferation (by the criterion of cells distribution over cell cycle). The results obtained, along with the critical analysis of published data, indicate that the ODC enzyme is involved in biochemical adaptation of mammals to natural and artificial hypobiosis. A decline in the ODC enzymatic activity indicates a decline in proliferative, functional, and metabolic activity of organs and tissues of mammals (bone marrow, mucosa of small intestine, thymus, spleen, neocortex, liver, kidneys) when entering the state of hypobiosis.

Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

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Glass, J.R.; Gerner, E.W.

1987-01-01

The mechanism of spermidine-induced ornithine decarboxylase (OCD, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine. Treatment of cells with 10 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [ 35 S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 0 C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 0 C for 3 hours with 10 μ M spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents had no effect of ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1 Improves Recombinant Protein Expression in the HEK293 Cell Line

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Laura Abaandou

2018-06-01

Full Text Available Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA and small interfering RNA (siRNA, to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1 gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293 cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

Trypanosoma cruzi Coexpressing Ornithine Decarboxylase and Green Fluorescence Proteins as a Tool to Study the Role of Polyamines in Chagas Disease Pathology

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Jeremías José Barclay

2011-01-01

Full Text Available Polyamines are essential for Trypanosoma cruzi, the causative agent of Chagas disease. As T. cruzi behaves as a natural auxotrophic organism, it relies on host polyamines biosynthesis. In this paper we obtained a double-transfected T. cruzi parasite that expresses the green fluorescent protein (GFP and a heterologous ornithine decarboxylase (ODC, used itself as a novel selectable marker. These autotrophic and fluorescent parasites were characterized; the ODC presented an apparent Km for ornithine of 0.51 ± 0.16 mM and an estimated Vmax value of 476.2 nmoles/h/mg of protein. These expressing ODC parasites showed higher metacyclogenesis capacity than the auxotrophic counterpart, supporting the idea that polyamines are engaged in this process. This double-transfected T. cruzi parasite results in a powerful tool—easy to follow by its fluorescence—to study the role of polyamines in Chagas disease pathology and in related processes such as parasite survival, invasion, proliferation, metacyclogenesis, and tissue spreading.

Ornithine Decarboxylase-Mediated Production of Putrescine Influences Ganoderic Acid Biosynthesis by Regulating Reactive Oxygen Species in Ganoderma lucidum.

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Wu, Chen-Gao; Tian, Jia-Long; Liu, Rui; Cao, Peng-Fei; Zhang, Tian-Jun; Ren, Ang; Shi, Liang; Zhao, Ming-Wen

2017-10-15

Putrescine is an important polyamine that participates in a variety of stress responses. Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the biosynthesis of putrescine. A homolog of the gene encoding ODC was cloned from Ganoderma lucidum In the ODC -silenced strains, the transcript levels of the ODC gene and the putrescine content were significantly decreased. The ODC -silenced strains were more sensitive to oxidative stress. The content of ganoderic acid was increased by approximately 43 to 46% in the ODC -silenced strains. The content of ganoderic acid could be recovered after the addition of exogenous putrescine. Additionally, the content of reactive oxygen species (ROS) was significantly increased by approximately 1.3-fold in the ODC -silenced strains. The ROS content was significantly reduced after the addition of exogenous putrescine. The gene transcript levels and the activities of four major antioxidant enzymes were measured to further explore the effect of putrescine on the intracellular ROS levels. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes. IMPORTANCE It is well known that ODC and the ODC-mediated production of putrescine play an important role in resisting various environmental stresses, but there are few reports regarding the mechanisms underlying the effect of putrescine on secondary metabolism in microorganisms, particularly in fungi. G. lucidum is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, a homolog of the gene encoding ODC was cloned in Ganoderma lucidum We found that the transcript level of the ODC gene and the content of putrescine were significantly decreased in the ODC -silenced strains. The content of

Differential retinoic acid inhibition of ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate and by germicidal ultraviolet light

International Nuclear Information System (INIS)

Lichti, U.; Patterson, E.; Hennings, H.; Yuspa, S.H.

1981-01-01

Several retinoids including retinoic acid effectively inhibit phorbol ester-mediated tumor promotion and ornithine decarboxylase (ODC) induction in mouse epidermis. To understand better the possible cellular site of action of retinoids, the inhibitory action of retinoic acid on the induction of ODC was compared for two distinctly different inducers, namely, 12-O-tetradecanoylphorbol-13-acetate (TPA) and germicidal ultraviolet light (uv), in primary mouse epidermal cell cultures. It was found that the induction of ODC by TPA is almost completely prevented by retinoic acid while the induction by uv is only moderately inhibited. The differential inhibition of enzyme induction cannot be accounted for by selective retinoid inhibition of DNA, RNA, or protein synthesis either alone or in concert with TPA or uv. These agents possibly act at transcription or translation, both of which are required for ODC induction by TPA or uv

Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in Vivo

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Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellet

1995-01-01

m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 ug of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks...

Ornithine decarboxylase activity in rat organs and tissues under artificial hypobiosis.

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Aksyonova, G E; Logvinovich, O S; Fialkovskaya, L A; Afanasyev, V N; Ignat'ev, D A; Kolomiytseva, I K

2010-09-01

The influence of hypothermia-hypoxia-hypercapnia on ornithine decarboxylase (ODC, EC 4.1.1.17) activities in rat organs and tissues and also on the thymocyte distribution throughout the cell cycle stages was studied. The state of artificial hypobiosis in rats on decrease in the body temperature to 14.4-18.0°C during 3.0-3.5 h was accompanied by drops in the ODC activities in the neocortex and liver by 50-60% and in rapidly proliferating tissues (thymus, spleen, and small intestine mucosa) by 80% of the control value. In kidneys the ODC activity raised to 200% of the control level. Twenty-four hours after termination of the cooling and replacing the rats under the standard conditions, the ODC activities in the neocortex, liver, kidneys, spleen, and intestinal mucosa returned to the control values, but remained decreased in the thymus. Forty-eight hours later the ODC activities in the thymus and spleen exceeded the normal level. The distribution of thymocytes throughout the cell cycle stages did not change in rats in the state of hypothermia (hypobiosis); 24 and 48 h after termination of the cooling the fraction of thymocytes in the S stage was decreased and the fraction of the cells in the G(0)+G(1) stage was increased. The normal distribution of thymocytes throughout the cell cycle stages recovered in 72 h. Thus, in the thymus the diminution of the ODC activity preceded the suppression of the cell proliferation rate. The tissue-specific changes in the ODC activity are suggested to reflect adaptive changes in the functional and proliferative activities of organs and tissues during the development of hypobiosis under conditions of hypothermia-hypoxia-hypercapnia.

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

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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.

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

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Julie P M Viala

Full Text Available 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.

Recovery from inhibition by UV-irradiation of ornithine decarboxylase induction in human cells: implication of excision repair

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Ben-Hur, E.; Prager, A. (Nuclear Research Centre-Negev, Beer-Sheva (Israel)); Buonaguro, F. (Argonne National Lab., IL (USA))

1982-05-01

Exposure of stationary-phase human breast carcinoma (T-47D) cells to far-UV light (254nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (Dsub(q)=2Jm/sup -2/) followed by an exponential decline (D/sub 0/=4.2Jm/sup -2/). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for 20-24h after irradiation. Hydroxyurea (HU) when present at 3mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50..mu..M of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, it is suggested that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.

Comparison of the effects of an ornithine decarboxylase inhibitor on the intestinal epithelium and on intestinal tumors.

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Tutton, P J; Barkla, D H

1986-12-01

Ornithine decarboxylase (ODC) catalyzes the rate-limiting step in the synthesis of polyamines, it has a short half-life, and its synthesis is under hormonal control. Recently, insight into the role of ODC and thus into the physiology of polyamines has been gained by the use of an inhibitor of ODC, difluoromethylornithine (DFMO). In the present report cell proliferation was measured by a stathmokinetic method in the crypt epithelium of the jejunum and colon of normal rats and in dimethylhydrazine-induced colonic tumors. Growth of human colon tumor xenografts in immunosuppressed mice and mouse colon tumor isografts was also assessed. Cell proliferation in primary colonic tumors was substantially suppressed by a single dose of DFMO at 100 mg/kg whereas the normal crypt epithelium of the small and large intestine required two doses at 400 mg/kg to produce a similar magnitude of inhibition of cell proliferation. DFMO was also found to suppress cell proliferation in, and the growth of, the transplantable colon cancers. Because of the apparent selectivity of the antimitotic activity of DFMO towards tumors, ODC inhibitors may prove to be useful anticancer drugs.

Regulation of polyamine synthesis in plants. Final progress report, July 1, 1991--December 31, 1994

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Malmberg, R.L.

1995-07-01

This research focused on unusual post-translational modifications occuring in a arginine decarboxylase cDNA clone in oats. A novel regulatory mechanism for polyamines was explored and an attempt was made to characterize it. A plant ornithine decarboxylase cDNA was identified in Arabidopsis. Further work remains on the mechanisms of polyamine regulation and function in plants.

Gamma radiation inhibits the appearance of induced ornithine decarboxylase activity in Chinese hamster cells

International Nuclear Information System (INIS)

Ben-Hur, E.; Heimer, Y.M.; Riklis, E.

1981-01-01

Ornithine decarboxylase activity of Chinese hamster cells (ODC, EC 4.1.1.17) can be induced in plateau phase by change of medium. Exposure of the cells to gamma radiation before induction reduces the amount of ODC activity induced. The dose-response curve is exponential with a D 0 of 106 krad. Exposure of BUdR-substituted cells is more effective in reducing ODC induction at high doses, with a D 0 of 38 krad. Cells can recover from the reduction incurred by 74 krad if enzyme induction is delayed for 2 hours after exposure. Treatment of the cells with psoralen-plus-light completely inhibits RNA synthesis without affecting protein synthesis (Heimer, Ben-Hur and Riklis 1977, 1978). Using this procedure it is shown that the effect of gamma radiation on inducible ODC activity is due not only to DNA damage but also involves a post-transcriptional effect. This conclusion is supported by employing a heat shock to inhibit protein synthesis prior to gamma-irradiation of log-phase cells. In such cells the increased activity of ODC upon transfer to 37 0 C is due primarily to enzyme synthesis using pre-existing RNA species during the first few hours. A low concentration of actinomycin D, which inhibits rRNA synthesis, applied during the recovery period, prevents the recovery of the cells' capacity for maximal ODC induction. This may indicate that, in order to recover, the cells have to repair damage to the ribosomes as well as to DNA. (author)

Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

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Daisuke Yamamoto

2010-09-01

Full Text Available Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

Cyclobutane-type pyrimidine photodimer formation and induction of ornithine decarboxylase in human skin fibroblasts after UV irradiation

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Niggli, H.J.; Roethlisberger, R.

1988-01-01

Cyclobutane-type pyrimidine photodimers as well as the induction of ornithine decarboxylase (ODC) may serve as biochemical markers of the mutagenic and carcinogenic effects of ultraviolet light (UV). For this reason, it is important to compare the formation of pyrimidine dimers with the induction of ODC in human skin fibroblasts after irradiation with UVC (200-290 nm) and UVB (290-320 nm). In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer yields (T-T) by high-pressure liquid chromatography in cultures of neonatal normal human foreskin-derived fibroblasts after irradiation with UVC and UVB light. It was found that the yield of dimerization and the ratio of T-T/C-T decreased from the UVC to the UVB region. Time-course studies of ODC-induction in the same cells indicated that the maximal activity after UVB irradiation was retarded compared to UVC exposure. For the UV-induced ODC-levels, however, no significant difference in maximal induction could be measured after UVC and UVB irradiation at fluences where comparable yields of thymine dimerization are produced. Similar ODC-maxima were obtained with strains from children, while cells from adults showed significantly less pronounced ODC induction, indicating that ODC-response decreases with age and may therefore be used as a marker of aging

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

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Park, Jae Hee; Lee, Chang Ki [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Oral Cancer Research Institute, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Hwang, Young Sun [Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Park, Kwang-Kyun [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Chung, Won-Yoon [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of)], E-mail: wychung@yuhs.ac

2008-07-03

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H{sub 2}O{sub 2} formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-{kappa}B) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-{kappa}B activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-{kappa}B signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

International Nuclear Information System (INIS)

Park, Jae Hee; Lee, Chang Ki; Hwang, Young Sun; Park, Kwang-Kyun; Chung, Won-Yoon

2008-01-01

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H 2 O 2 formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-κB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-κB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent

Polyamine and amino acid content, and activity of polyamine-synthesizing decarboxylases, in liver of streptozotocin-induced diabetic and insulin-treated diabetic rats

OpenAIRE

Brosnan, Margaret E.; Roebothan, Barbara V.; Hall, Douglas E.

1980-01-01

1. Concentrations of polyamines, amino acids, glycogen, nucleic acids and protein, and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, were measured in livers from control, streptozotocin-diabetic and insulin-treated diabetic rats. 2. Total DNA per liver and protein per mg of DNA were unaffected by diabetes, whereas RNA per mg of DNA and glycogen per g of liver were decreased. Insulin treatment of diabetic rats induced both hypertrophy and hyperplasia, as indicat...

Arginase and Arginine Decarboxylase - Where Do the Putative Gate Keepers of Polyamine Synthesis Reside in Rat Brain?

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Daniela Peters

Full Text Available Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine

Growth inhibition of human breast cancer cells and down-regulation of ODC1 and ADA genes by Nepeta binaloudensis

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Akbar Safipour Afshar

Full Text Available ABSTRACT Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231 compared to non-cancer line (MCF-10A. The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

Screening method for detection of immediate amino acid decarboxylases--producing bacteria implicated in food poisoning.

Science.gov (United States)

Hussain, Husniza; Mohd Fuat, A R; Vimala, B; Ghazali, H M

2011-08-01

Assessment of amino acid decarboxylase activity can be conducted using tubed broth or plated agar. In this study, the test was carried out in microtitre plates containing lysine, ornithine, arginine, tyrosine, tryptophan, phenylalanine or histidine as biogenic amine precursors. Møller decarboxylase base broth (MDB) with or without 1% of a known amino acid were added to wells of a 96 well-microtitre plate. The wells were inoculated with Escherichia coli, Klebsiella pneumoniae, Acinetobacter anitratus or Staphylococcus aureus to the final concentration of 6.0 x 10(7) cfu/ml and incubated at 35ºC. The absorbance of the culture broth was read at 570 nm at 0, 1.0, 2.0, 3.0, 4.0, 5.5, 6.5 and 7.5 hour. Comparison of means of A'(570) between 0 hour and a specified incubation time was determined statistically. Positive decarboxylase activities were detected in the media inoculated with E. coli and K. pneumoniae in less than 6 hours. The current method is suitable for immediate producers of amino acid decarboxylase enzymes. It costs less as it uses less amino acid and it has the potential to be used for screening aliquots of food materials for amino acid decarboxylase activities.

High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.

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Reddy, S G; Cochran, B J; Worth, L L; Knutson, V P; Haddox, M K

1994-04-01

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

Diethylglyoxal bis(guanylhydrazone): a novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use.

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Elo, H; Mutikainen, I; Alhonen-Hongisto, L; Laine, R; Jänne, J

1988-07-01

Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied (Ki approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

Ornithine transport and exchange in Streptococcus lactis

International Nuclear Information System (INIS)

Thompson, J.

1987-01-01

Resting cells of Streptococcus lactis 133 appeared to accumulate [ 14 C]ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular [ 14 C]ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular [ 14 C]ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exist of [ 14 C]ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and new decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of [ 14 C]ornithine have been examined. The data suggest that common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis

Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri

DEFF Research Database (Denmark)

Møller, Kasper; Langkjær, Rikke Breinhold; Nielsen, Jens

2004-01-01

was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts...... is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae...

MicroRNA regulation in Ames dwarf mouse liver may contribute to delayed aging.

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Bates, David J; Li, Na; Liang, Ruqiang; Sarojini, Harshini; An, Jin; Masternak, Michal M; Bartke, Andrzej; Wang, Eugenia

2010-02-01

The Ames dwarf mouse is well known for its remarkable propensity to delay the onset of aging. Although significant advances have been made demonstrating that this aging phenotype results primarily from an endocrine imbalance, the post-transcriptional regulation of gene expression and its impact on longevity remains to be explored. Towards this end, we present the first comprehensive study by microRNA (miRNA) microarray screening to identify dwarf-specific lead miRNAs, and investigate their roles as pivotal molecular regulators directing the long-lived phenotype. Mapping the signature miRNAs to the inversely expressed putative target genes, followed by in situ immunohistochemical staining and in vitro correlation assays, reveals that dwarf mice post-transcriptionally regulate key proteins of intermediate metabolism, most importantly the biosynthetic pathway involving ornithine decarboxylase and spermidine synthase. Functional assays using 3'-untranslated region reporter constructs in co-transfection experiments confirm that miRNA-27a indeed suppresses the expression of both of these proteins, marking them as probable targets of this miRNA in vivo. Moreover, the putative repressed action of this miRNA on ornithine decarboxylase is identified in dwarf mouse liver as early as 2 months of age. Taken together, our results show that among the altered aspects of intermediate metabolism detected in the dwarf mouse liver--glutathione metabolism, the urea cycle and polyamine biosynthesis--miRNA-27a is a key post-transcriptional control. Furthermore, compared to its normal siblings, the dwarf mouse exhibits a head start in regulating these pathways to control their normality, which may ultimately contribute to its extended health-span and longevity.

Characterization of arginine decarboxylase from Dianthus caryophyllus.

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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%.

Serum levels of polyamine synthesis enzymes increase in diabetic patients with breast cancer

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V Kenan Çelik

2017-09-01

Full Text Available In this study, it was aimed to investigate the relationship between diabetes and breast cancer and the detection of enzymes and ornithine levels in polyamine synthesis pathway in diabetes, breast cancer and diabetic breast cancer patients. Methods: Ornithine, arginine decarboxylase, ornithine decarboxylase and agmatinase levels have been measured in serum of all groups. Ornithine levels were measured spectrophotometrically. Arginine decarboxylase, ornithine decarboxylase and agmatinase levels were determined by ELISA kits. Results: Except for the diabetic group, the levels of enzymes in the polyamine synthesis pathway were increased in all and statistically significant (P < 0.05. The increase in the levels of agmatinase was very important among the enzymes (P < 0.001. Conclusions: Decreased levels of polyamine synthase enzymes in diabetes mellitus were found to be increased patients with breast cancer. Whether and how diabetes-based breast cancer development relates to increase activity of enzymes responsible for polyamine synthesis requires further mechanistic and prospective monitoring studies in larger patient cohorts.

Antepartum Ornithine Transcarbamylase Deficiency

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Hitoshi Nakajima

2014-11-01

Full Text Available Ornithine transcarbamylase deficiency (OTCD is the most common type urea cycle enzyme deficiencies. This syndrome results from a deficiency of the mitochondrial enzyme ornithine transcarbamylase, which catalyzes the conversion of ornithine and carbamoyl phosphate to citrullin. Our case was a 28-year-old female diagnosed with OTCD following neurocognitive deficit during her first pregnancy. Although hyperammonemia was suspected as the cause of the patient's mental changes, there was no evidence of chronic liver disease. Plasma amino acid and urine organic acid analysis revealed OTCD. After combined modality treatment with arginine, sodium benzoate and hemodialysis, the patient's plasma ammonia level stabilized and her mental status returned to normal. At last she recovered without any damage left.

Differential effects of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the testosterone-induced growth of ventral prostate and seminal vesicles of castrated rats.

OpenAIRE

Käpyaho, K; Kallio, A; Jänne, J

1984-01-01

2-Difluoromethylornithine totally prevented any increases in putrescine and spermidine concentrations in the ventral prostate of castrated rats during a 6-day testosterone treatment. Prostatic ornithine decarboxylase activity was inhibited by 80%, whereas S-adenosylmethionine decarboxylase was stimulated by more than 9-fold. In seminal vesicle, the inhibition of putrescine and spermidine accumulation, as well as of ornithine decarboxylase activity, was only minimal, and no stimulation of S-ad...

Characterization of OAZ1 and its potential functions in goose follicular development

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Bo Kang

2017-03-01

Conclusions: The goose OAZ1 structure confirms that OAZ1 plays an important role in ornithine decarboxylase-mediated regulation of polyamine homeostasis. Our findings provide an evidence for a potential function of OAZ1 in follicular development, ovulation and regression.

The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

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Kyriakidis Dimitrios A

2004-06-01

Full Text Available Abstract This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the

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

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Gloria E Hoffman

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

Differential effects of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the testosterone-induced growth of ventral prostate and seminal vesicles of castrated rats.

Science.gov (United States)

Käpyaho, K; Kallio, A; Jänne, J

1984-05-01

2-Difluoromethylornithine totally prevented any increases in putrescine and spermidine concentrations in the ventral prostate of castrated rats during a 6-day testosterone treatment. Prostatic ornithine decarboxylase activity was inhibited by 80%, whereas S-adenosylmethionine decarboxylase was stimulated by more than 9-fold. In seminal vesicle, the inhibition of putrescine and spermidine accumulation, as well as of ornithine decarboxylase activity, was only minimal, and no stimulation of S-adenosylmethionine decarboxylase was observed. Administration of methylglyoxal bis(guanylhydrazone) to castrated androgen-treated rats resulted in a marked increase in concentrations of all prostatic polyamines. Prostatic ornithine decarboxylase activity was nearly 2 times and adenosylmethionine decarboxylase activity 9 times higher than that of the testosterone-treated animals. In contrast with ventral prostate, methylglyoxal bis(guanylhydrazone) treatment inhibited moderately the accumulation of spermidine and spermine in seminal vesicle, although both ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were stimulated. Difluoromethylornithine inhibited significantly the weight gain of ventral prostate, but methylglyoxal bis(guanylhydrazone) produced a substantial increase in prostatic weight. These changes were largely due to the fact that the volume of prostatic secretion was greatly decreased by difluoromethylornithine, whereas methylglyoxal bis(guanylhydrazone) increased the amount of secretion. Treatment with difluoromethylornithine strikingly increased the methylglyoxal bis(guanylhydrazone) content of both ventral prostate and seminal vesicle, but even under these conditions the drug concentration remained low in comparison with other tissues. The results indicate that a combined use of these two polyamine anti-metabolites does not necessarily result in a synergistic growth inhibition of the androgen-induced growth of male accessory sexual glands.

In vivo trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

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Bacchi, C J; Brun, R; Croft, S L; Alicea, K; Bühler, Y

1996-01-01

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for trypanocidal activities in human and veterinary trypanosomes of African origin. One agent, CGP 40215A, a bicyclic analog of MGBG which also resembles the diamidines diminazene (Berenil) and pentamidine, was curative of infections by 19 isolates of Trypanosoma brucei subspecies as well as a Trypanosoma congolense isolate. Several of these isolates were resistant to standard trypanocides. Curative doses were < or = 25 mg/kg of body weight/day for 3 days in these acute laboratory model infections. In addition, CGP 40215A also cured a model central nervous system infection in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl, eflornithine). Curative combinations were 14 days of oral 2% DFMO (approximately 5 g/kg/day) plus 5, 10, or 25 mg/kg/day for 3 or 7 days given by intraperitoneal injection or with a miniosmotic pump. Combinations were most effective if CGP 40215A was given in the second half or at the end of the DFMO regimen. MGBG has modest activity as an inhibitor of trypanosome S-adenosylmethionine decarboxylase (50% inhibitory concentration [IC50]. 130 microM), while CGP 40215A was a more active inhibitor (IC50, 20 microM). Preincubation of trypanosomes with CGP 40215A for 1 h caused a reduction in spermidine content (36%) and an increase in putrescine content (20%), indicating that one possible mechanism of its action may be inhibition of polyamine biosynthesis. PMID:8726018

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

Science.gov (United States)

Shu, Qunfeng; Xu, Meijuan; Li, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong; Rao, Zhiming

2018-05-04

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

AmcA - a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis

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Lukas eSchafferer

2015-04-01

Full Text Available Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine. In this study, we demonstrate a role of the putative mitochondrial transporter AmcA (AFUA_8G02760 in siderophore biosynthesis of A. fumigatus.Consistent with a role in cellular ornithine handling, AmcA-deficiency resulted in decreased cellular ornithine and arginine contents as well as decreased siderophore production on medium containing glutamine as the sole nitrogen source. In support, arginine and ornithine as nitrogen sources did not impact siderophore biosynthesis due to cytosolic ornithine availability. As revealed by Northern blot analysis, transcript levels of siderophore biosynthetic genes were unresponsive to the cellular ornithine level. In contrast to siderophore production, AmcA deficiency did only mildly decrease the cellular polyamine content, demonstrating cellular prioritization of ornithine use. Nevertheless, AmcA-deficiency increased the susceptibility of A. fumigatus to the polyamine biosynthesis inhibitor eflornithine, most likely due to the decreased ornithine pool. AmcA-deficiency decreased the growth rate particularly on ornithine as the sole nitrogen source during iron starvation and sufficiency, indicating an additional role in the metabolism and fitness of A. fumigatus, possibly in mitochondrial ornithine import. In the Galleria mellonella infection model, AmcA-deficiency did not affect virulence of A. fumigatus, most likely due to the residual siderophore production and arginine availability in this host niche.

L-ornithine-L-aspartate infusion efficacy in hepatic encephalopathy

International Nuclear Information System (INIS)

Ahmad, I.

2008-01-01

To determine the efficacy of L-ornithine-L-aspartate in treatment of hepatic encephalopathy. Cirrhotic patients with hyperammonemia and overt hepatic encephalopathy were enrolled. Eighty patients were randomized to two treatment groups, L-ornithine-L-aspartate (20g/d) or placebo, both dissolved in 250mL of 5% dextrose water and infused intravenously for four hours a day for five consecutive days with 0.5 g/kg dietary protein intake at the end of daily treatment period. Outcome variables were postprandial blood ammonia and mental state grade. Adverse reactions and mortality were also determined. Both treatment groups were comparable regarding age, gender, etiology of cirrhosis, Child-Pugh class, mental state grade and blood ammonia at baseline. Although, improvement occurred in both groups, there was a greater improvement in L-ornithine-L-aspartate group with regard to both variables. Four patients in the placebo group and 2 in L-ornithine-L-aspartate group died. L-ornithine-L-aspartate infusions were found to be effective in cirrhotic patients with hepatic encephalopathy. (author)

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

Science.gov (United States)

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Molecular mechanisms underlying Grateloupia imbricata (Rhodophyta) carposporogenesis induced by methyl jasmonate.

Science.gov (United States)

Garcia-Jimenez, Pilar; Montero-Fernández, Montserrat; Robaina, Rafael R

2017-12-01

When applied in vitro, methyl jasmonate is sensed by the red seaweed Grateloupia imbricate, substantially and visually affecting its carposporogenesis. However, although there is some understanding of the morphological changes induced by methyl jasmonate in vitro, little is known about the genes that are involved in red seaweed carposporogenesis and how their protein products act. For the work reported herein, the expression of genes in red seaweed that encode enzymes involved in the synthesis of methyl jasmonate (jasmonic acid carboxyl methyl transferase and a putative methyl transferase) was monitored. Additionally the genes involved in oxidation (cytochrome P450 and WD40), jasmonate synthesis, signal transduction, and regulation of reactive oxygen species (MYB), and reproduction (ornithine decarboxylase) were monitored. To determine when or if the aforementioned genes were expressed during cystocarp development, fertilized and fertile thalli were exposed to methyl jasmonate and gene expression was measured after 24 and 48 h. The results showed that methyl jasmonate promoted differential gene expression in fertilized thalli by 24 h and upregulated expression of the ornithine decarboxylase gene only by 48 h in fertile thalli (0.75 ± 003 copies · μL -1 at 24 h vs. 1.11 ± 0.04 copies · μL -1 at 48 h). We conclude that Ornithine decarboxylase expression involves methyl jasmonate signaling as well as development and maturation of cystocarps. © 2017 Phycological Society of America.

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

Science.gov (United States)

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

2016-03-29

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.

Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism

Science.gov (United States)

Weinstein, L. H.; Osmeloski, J. F.; Wettlaufer, S. H.; Galston, A. W.

1987-01-01

In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.

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

Science.gov (United States)

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 glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

A systematic review on aromatic L-amino acid decarboxylase (5-hydroxytryptophan decarboxylase)

International Nuclear Information System (INIS)

Rahman, M.K.; Nagatsu, T.

1988-11-01

Aromatic L-amino acid decarboxylase (AADC, EC. 4.1.1.28) with L-5-hydroxytryptophan as a substrate (also called L-5-hydroxytryptophan decarboxylase, 5-HTPDC) decarboxylates L-5-hydroxytryptophan to serotonin (5-HT), an important neurotransmitter that involved in the regulation of neuronal functions, behaviour and emotion of higher animals. As it is an important enzyme, many researchers are now working on its physiological functions and properties and also on its isolation, purification and characterization from mammalian tissues. But up to now no systematic review studies have been done on this enzyme. We made systematic studies on this enzyme in tissues and brains of rats, and human subjects. We also developed highly sensitive assay methods of the enzyme. This new method led us to discover the enzyme in the sera of various animals. We examined the developmental changes of 5-HTPDC in the sera of animals. We discovered an endogenous inhibitor of the enzyme in the monkey blood. The purification of the enzyme were performed by us and other researches from the sera, brains, adrenals, liver and kidneys of mammals. These and other results of up to date research papers on 5-HTPDC have been reviewed in this paper. (author). 71 refs, 10 figs, 14 tabs

GROWTH-INHIBITION OF 2 SOLID TUMORS IN MICE, CAUSED BY POLYAMINE DEPLETION, IS NOT ATTENDED BY ALTERATIONS IN CELL-CYCLE PHASE DISTRIBUTION

NARCIS (Netherlands)

HESSELS, J; KINGMA, AW; MUSKIET, FAJ; SARHAN, S; SEILER, N

1991-01-01

We studied the effect of polyamine depletion on growth and cell cycle characteristics of subcutaneously grown Lewis lung carcinoma (LLC) and fibrosarcoma (FIO 26) in mice. Polyamine depletion was achieved by inhibition of ornithine decarboxylase using 2-(difluoromethyl)ornithine, limitation of

The Combined Effect of Caffeine and Ornithine on the Mood of Healthy Office Workers

Science.gov (United States)

Misaizu, Akane; Kokubo, Takeshi; Tazumi, Kyoko; Kanayama, Masaya; Miura, Yutaka

2014-01-01

Caffeine is widely consumed and well known for stimulating the central nervous system. When developing new foods and beverages that contain caffeine, it is important to explore the potential synergistic effects of consuming amino acids and other food ingredients with caffeine on humans. Given the physiological pathways affected by the amino acid ornithine, consumption of ornithine with caffeine may have synergistic effects. The purpose of the present study was to examine the effect of consuming caffeine with ornithine in humans. The study used a randomized, placebo-controlled, double-blinded crossover design. The subjects were all healthy office workers who ingested the placebo, 100 mg caffeine, or 100 mg caffeine plus 200 mg ornithine in the morning and completed questionnaires about their mood. Office workers who consumed the combination of caffeine and ornithine had higher mood ratings 8 h after consumption than office workers who consumed caffeine alone. The results of the present study suggest that there is a unique synergistic effect between caffeine and ornithine on the mood of healthy office workers and that ornithine may potentiate the effects of caffeine. PMID:25580405

Radiometric microassay for glutamic acid decarboxylase

Energy Technology Data Exchange (ETDEWEB)

Maderdrut, J L [North Carolina Dept. of Mental Health, Raleigh (USA); North Carolina Univ., Chapel Hill (USA). School of Medicine)

1979-01-01

A simple method for purifying L-(/sup 3/H) glutamic acid and incubation conditions suitable for estimating L-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedure for separating ..gamma..-aminobutyric acid from L-glutamate are outlined and compared. Recycling increases the sensitivity of the cation-exchange method by 6-7 fold. L-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 ..mu..g. The cation-exchange method is compared with the anion-exchange and CO/sub 2/-trapping methods. L-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 21/4 (stage 14) using the cation-exchange method. This is 5-6 days earlier than L-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. L-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis.

Interorgan metabolism of ornithine phenylacetate (OP)-A novel strategy for treatment of hyperammonemia

DEFF Research Database (Denmark)

Dadsetan, Sherry; Sørensen, Michael; Bak, Lasse Kristoffer

2013-01-01

Combined administration of ornithine and phenylacetate (OP) is proposed as a novel treatment of hyperammonemia and hepatic encephalopathy. Ornithine is believed to increase ammonia fixation into glutamine in muscle tissue and glutamine is subsequently thought to react with phenylacetate forming......, skeletal muscle, liver and kidney. In BDL rats, OP treatment reduced arterial ammonia concentration and increased that of glutamine 30min after the treatment but not after 15h. OP treatment did not increase (15)N labeling in glutamine from [2,5-(15)N]ornithine and (15)NH(4)(+) in skeletal muscle or liver....... However, the extent of glutamine labeling from [2,5-(15)N]ornithine or (15)NH(4)(+) was similar in arterial blood and liver and higher than that in skeletal muscle. These findings suggest that the effect of OP was related to hepatic metabolism of ornithine. PAGN could not be detected in urine or blood...

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Czech Academy of Sciences Publication Activity Database

Cvikrová, Milena; Gemperlová, Lenka; Eder, Josef; Zažímalová, Eva

2008-01-01

Roč. 27, č. 7 (2008), s. 1147-1156 ISSN 0721-7714 R&D Projects: GA AV ČR IAA6038303 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arginine decarboxylase * Diamine oxidase * Ornithine decarboxylase Subject RIV: ED - Physiology Impact factor: 1.946, year: 2008

Improved tolerance of abdominal large-volume radiotherapy due to ornithine aspartate

International Nuclear Information System (INIS)

Kuttig, H.

1983-01-01

The influence of ornithine aspartate on supporting the hepatic function was investigated in a group of 47 patients with tumour dissemination in the pelvic and abdominal region, randomised on the basis of the progress of the serum enzymes GOT, GPT, LAD, LDH, LAP and the alkaline phosphatase during and following completion of a course of large-volume radiotherapy. The adjuvant therapy with ornithine aspartate resulted in reduced enzyme movement with an earlier tendency to normalisation. The results, which are borne out by statistics, clearly show an improvement in the hepatic function on detoxication of toxic degradation products of radiotherapy with reduced impairment of the body's own defence mechanisms. Subjectively too, the course of treatment with ornithine aspartate showed a reduced ratio of side effects as regards lassitude and impairment of the patient's general well-being as compared with the group of patients to whom ornithine aspartate was not simultaneously administered. (orig.) [de

Chemical labeling of gluatmate decarboxylase in vivo

International Nuclear Information System (INIS)

Rando, R.R.

1981-01-01

Mouse brain glutamate decarboxylase(s) was specifically titrated in vivo and in crude brain homogenates by a combination of gabaculine and [alpha-3H]acetylenic gamma-aminobutyric acid. This specific titration is based on the differential spectra of action of these two mechanism-based enzyme inactivators. The specificity of the titration in vitro was demonstrated by showing that the time course of radioactivity incorporation exactly paralleled the time course for glutamate of decarboxylase inactivation. This means that there is approximately 0.66 nmol of glutamate decarboxylase/0.5 g of mouse brain, assuming the stoichiometry of inactivator bound to enzyme is one. This value is similar to the one obtained from a calculation based on the enzyme purification data

A radiometric microassay for glutamic acid decarboxylase

International Nuclear Information System (INIS)

Maderdrut, J.L.; North Carolina Univ., Chapel Hill

1979-01-01

A simple method for purifying L-[ 3 H] glutamic acid and incubation conditions suitable for estimating L-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedure for separating γ-aminobutyric acid from L-glutamate are outlined and compared. Recycling increases the sensitivity of the cation-exchange method by 6-7 fold. L-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 μg. The cation-exchange method is compared with the anion-exchange and CO 2 -trapping methods. L-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 21/4 (stage 14) using the cation-exchange method. This is 5-6 days earlier than L-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. L-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis. (author)

Genetics Home Reference: ornithine transcarbamylase deficiency

Science.gov (United States)

... belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions ... Baby's First Test GeneReview: Ornithine Transcarbamylase Deficiency GeneReview: Urea Cycle Disorders Overview MedlinePlus Encyclopedia: Hereditary urea cycle abnormality National ...

Central L-ornithine, but not polyamines, induces a hypnotic effect in neonatal chicks under acute stress.

Science.gov (United States)

Kurauchi, Isao; Shigemi, Kazutaka; Kabuki, Yusuke; Hamasu, Kousuke; Yamane, Haruka; Aoki, Mami; Kawada, Yoko; Morishita, Koji; Denbow, D Michael; Furuse, Mitsuhiro

2010-02-01

To clarify whether L-ornithine and/or its metabolite involves sedative and hypnotic effects under social separation stress, the effects of intracerebroventricular (i.c.v.) injection of L-ornithine and polyamines (putrescine, spermidine and spermine) were compared in chicks. Birds were injected i.c.v. with 0.5 mumol of L-ornithine, putrescine, spermidine, spermine or saline (control). After injection, chicks were immediately separated from the flock and monitored for the number of distress vocalizations and various postures. L-Ornithine greatly attenuated the stress response and caused sedative and hypnotic effects. Among the polyamines, only putrescine attenuated distress vocalizations but did not induce sleep. In conclusion, the sedative and hypnotic effect of L-ornithine was mainly induced by L-ornithine itself, while the polyamines contributed to the sedative, but not hypnotic, effect under social separation stress.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

Science.gov (United States)

Kumar, Ajay; Yellepeddi, Venkata K; Vangara, Kiran K; Strychar, Kevin B; Palakurthi, Srinath

2011-11-01

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Randomised controlled trial of the effects of L-ornithine on stress markers and sleep quality in healthy workers

OpenAIRE

Miyake, Mika; Kirisako, Takayoshi; Kokubo, Takeshi; Miura, Yutaka; Morishita, Koji; Okamura, Hisayoshi; Tsuda, Akira

2014-01-01

Background L-ornithine is a non-essential, non-protein amino acid. Although L-ornithine is contained in various foods, the amount is usually small. Recently, studies have shown that orally administered L-ornithine reduced the stress response in animals. From these findings, we speculated that L-ornithine may play a role in the relieve of stress and improve sleep and fatigue symptoms in humans. Through a randomised, double-blind, placebo-controlled clinical study, we asked if L-ornithine could...

Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

Science.gov (United States)

Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

2014-08-01

Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

Polyamines and plant stress - Activation of putrescine biosynthesis by osmotic shock

Science.gov (United States)

Flores, H. E.; Galston, A. W.

1982-01-01

The putrescine content of oat leaf cells and protoplasts increases up to 60-fold within 6 hours of exposure to osmotic stress (0.4 to 0.6 molar sorbitol). Barley, corn, wheat, and wild oat leaves show a similar response. Increased arginine decarboxylase activity parallels the rise in putrescine, whereas ornithine decarboxylase remains unchanged. DL-alpha-Difluoromethylarginine, a specific irreversible inhibitor of arginine decarboxylase, prevents the stress-induced rise in increase in arginine decarboxylase activity and putrescine synthesis, indicating the preferential activation of this pathway.

S-adenosylmethionine decarboxylase from baker's yeast.

Science.gov (United States)

Pösö, H; Sinervirta, R; Jänne, J

1975-01-01

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate. PMID:1108876

Evolutionary Profiling of Group II Pyridoxal-Phosphate-Dependent Decarboxylases Suggests Expansion and Functional Diversification of Histidine Decarboxylases in Tomato

Directory of Open Access Journals (Sweden)

Rahul Kumar

2016-03-01

Full Text Available Pyridoxal phosphate (PLP-dependent enzymes are one of the most important enzymes involved in plant N metabolism. Here, we explored the evolution of group II PLP-dependent decarboxylases (PLP_deC, including aromatic L-amino acid decarboxylase, glutamate decarboxylase, and histidine decarboxylase in the plant lineage. Gene identification analysis revealed a higher number of genes encoding PLP_deC in higher plants than in lower plants. Expression profiling of PLP_deC orthologs and syntelogs in (L. Heynh., pepper ( L., and tomato ( L. pointed toward conserved as well as distinct roles in developmental processes such as fruit maturation and ripening and abiotic stress responses. We further characterized a putative promoter of tomato ripening-associated gene ( operating in a complex regulatory circuit. Our analysis provides a firm basis for further in-depth exploration of the PLP_deC gene family, particularly in the economically important Solanaceae family.

Action of ornithine alpha-ketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects.

Science.gov (United States)

Cynober, L; Coudray-Lucas, C; de Bandt, J P; Guéchot, J; Aussel, C; Salvucci, M; Giboudeau, J

1990-02-01

Ornithine alpha-ketoglutarate (OKG) has been useful as an adjuvant of enteral and parenteral nutrition. However, its metabolism and mechanism of action remain unclear although it is known that alpha-ketoglutarate (alpha KG) and ornithine (ORN) follow, in part, common metabolic pathways. Six fasting healthy male subjects underwent three separate oral load tests: (i) they received 10 g of OKG (i.e., 3.6 g of alpha KG and 6.4 g of ORN); (ii) 6.4 g of ORN as ornithine hydrochloride, and (iii) 3.6 g of alpha KG as calcium alpha-ketoglutarate. Blood was drawn 15 times over a five-hour period for measurements of plasma amino acids, alpha KG, insulin, and glucagon. After OKG and ORN administration, plasma ORN peaked at 60-75 min (494 +/- 91 and 541 +/- 85 mumol/L). The increase in plasma alpha KG was very small. OKG, alpha KG, and ORN all increased glutamate concentrations at 60 min (mean: +43%, +68%, +68%, respectively, p less than 0.05 compared to basal values). However, only OKG increased proline and arginine levels at 60 min (mean: +35%, p less than 0.01 and mean: +41%, p less than 0.05). Furthermore, glutamate, proline, and arginine concentrations correlated linearly with ornithine levels at 60 min. Finally, OKG increased insulinemia and glucagonemia (mean: +24% at 15 min, p less than 0.05 and +30% at 60 min, p less than 0.01, respectively). These data provide evidence that the combination of ORN and alpha KG modifies amino acid metabolism in a way which is not observed when they are administered separately. In addition, the OKG-mediated increase in insulin levels probably does not appear to result from a direct action of ORN on pancreatic secretion.

L-ornithine derived polyamines in cystic fibrosis airways.

Directory of Open Access Journals (Sweden)

Hartmut Grasemann

Full Text Available Increased arginase activity contributes to airway nitric oxide (NO deficiency in cystic fibrosis (CF. Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.

Evidence for arginine as the endogenous precursor of necines in heliotropium.

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Birecka, H; Birecki, M; Frohlich, M W

1987-05-01

In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to (14)C-labeled CO(2) for 44 hours, the incorporation of (14)C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with alpha-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on (14)C incorporation into the necines. In contrast, alpha-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of (14)C into the necines of both species; the inhibitor did not affect the absolute incorporation of (14)C from exogenous [1,4-(14)C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-(14)C]arginine, but also exogenous l-[5-(14)C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of (14)C-labeled CO(2) assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed.

Evidence for Arginine as the Endogenous Precursor of Necines in Heliotropium1

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Birecka, Helena; Birecki, Mieczyslaw; Frohlich, M. W.

1987-01-01

In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to 14C-labeled CO2 for 44 hours, the incorporation of 14C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with α-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on 14C incorporation into the necines. In contrast, α-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of 14C into the necines of both species; the inhibitor did not affect the absolute incorporation of 14C from exogenous [1,4-14C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-14C]arginine, but also exogenous l-[5-14C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of 14C-labeled CO2 assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed. PMID:16665402

Randomised controlled trial of the effects of L-ornithine on stress markers and sleep quality in healthy workers.

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Miyake, Mika; Kirisako, Takayoshi; Kokubo, Takeshi; Miura, Yutaka; Morishita, Koji; Okamura, Hisayoshi; Tsuda, Akira

2014-06-03

L-ornithine is a non-essential, non-protein amino acid. Although L-ornithine is contained in various foods, the amount is usually small.Recently, studies have shown that orally administered L-ornithine reduced the stress response in animals.From these findings, we speculated that L-ornithine may play a role in the relieve of stress and improve sleep and fatigue symptoms in humans. Through a randomised, double-blind, placebo-controlled clinical study, we asked if L-ornithine could be beneficial to stress and sleep in healthy workers. Fifty-two apparently healthy Japanese adults who had previously felt slight stress as well as fatigue were recruited to be study participants and were randomly divided into either the L-ornithine (400 mg/day) or placebo group. They orally consumed the respective test substance every day for 8 weeks. Serum was collected for the assessment of cortisol and dehydroepiandrosterone-sulphate (DHEA-S). Perceived mood and quality of sleep were measured by the Profile of Mood States (POMS), Athens Insomnia Scale (AIS), and Ogri-Shirakawa-Azumi sleep inventory MA version (OSA-MA). Serum cortisol levels and the cortisol/DHEA-S ratio were significantly decreased in the L-ornithine group in comparison with the placebo group. Also, anger was reduced and perceived sleep quality was improved in the L-ornithine group. L-ornithine supplementation has the potential to relieve stress and improve sleep quality related to fatigue, both objectively and subjectively.

Role of Arginine decarboxylase (ADC) in Arabidopsis thaliana defence against the pathogenic bacterium Pseudomonas viridiflava.

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Rossi, F R; Marina, M; Pieckenstain, F L

2015-07-01

Polyamine biosynthesis starts with putrescine production through the decarboxylation of arginine or ornithine. In Arabidopsis thaliana, putrescine is synthesised exclusively by arginine decarboxylase (ADC), which exists as two isoforms (ADC1 and 2) that are differentially regulated by abiotic stimuli, but their role in defence against pathogens has not been studied in depth. This work analysed the participation of ADC in Arabidopsis defence against Pseudomonas viridiflava. ADC activity and expression, polyamine levels and bacterial resistance were analysed in null mutants of each ADC isoform. In non-infected wild-type (WT) plants, ADC2 expression was much higher than ADC1. Analysis of adc mutants demonstrated that ADC2 contributes to a much higher extent than ADC1 to basal ADC activity and putrescine biosynthesis. In addition, adc2 mutants showed increased basal expression of salicylic acid- and jasmonic acid-dependent PR genes. Bacterial infection induced putrescine accumulation and ADC1 expression in WT plants, but pathogen-induced putrescine accumulation was blocked in adc1 mutants. Results suggest a specific participation of ADC1 in defence, although basal resistance was not decreased by dysfunction of either of the two ADC genes. In addition, and as opposed to WT plants, bacterial infection increased ADC2 expression and ADC activity in adc1 mutants, which could counterbalance the lack of ADC1. Results demonstrate a major contribution of ADC2 to total ADC activity and the specific induction of ADC1 in response to infection. A certain degree of functional redundancy between the two isoforms in relation to their contribution to basal resistance is also evident. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

Functional characterization of an ornithine cyclodeaminase-like protein of Arabidopsis thaliana

OpenAIRE

Sharma, Sandeep; Shinde, Suhas; Verslues, Paul E

2013-01-01

Background In plants, proline synthesis occurs by two enzymatic steps starting from glutamate as a precursor. Some bacteria, including bacteria such as Agrobacterium rhizogenes have an Ornithine Cyclodeaminase (OCD) which can synthesize proline in a single step by deamination of ornithine. In A. rhizogenes, OCD is one of the genes transferred to the plant genome during the transformation process and plants expressing A. rhizogenes OCD have developmental phenotypes. One nuclear encoded gene of...

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism.

Science.gov (United States)

Khomutov, M A; Weisell, J; Hyvönen, M; Keinänen, T A; Vepsäläinen, J; Alhonen, L; Khomutov, A R; Kochetkov, S N

2013-12-01

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells "proinhibitor" of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.

MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency

NARCIS (Netherlands)

Sacksteder, K. A.; Morrell, J. C.; Wanders, R. J.; Matalon, R.; Gould, S. J.

1999-01-01

Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO(2). Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role

Effect of ornithine on ammonia utilization and urea synthesis in isolated hepatocytes from fed rats

International Nuclear Information System (INIS)

Garwacki, S.; Wiechetek, M.; Souffrant, W.B.

1988-01-01

The effect of ornithine on the ammonia utilization and urea synthesis in hepatocytes isolated from fed male Wistar rats was investigated. On the basis of the 15 N tracer technique, it was found that ornithine stimulated urea synthesis with an increased utilization of the exogenously marked ammonia for urea, but deminished its utilization in other N-metabolic processes. The results also showed that the stimulation of urea synthesis due to ornithine resulted from the utilization of both exogenous and endogenous sources. (author)

Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

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Chocklett, Samuel W; Sobrado, Pablo

2010-08-10

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

Contribution of polyamines metabolism and GABA shunt to chilling tolerance induced by nitric oxide in cold-stored banana fruit.

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Wang, Yansheng; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

2016-04-15

Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline. Copyright © 2015 Elsevier Ltd. All rights reserved.

Late-onset ornithine transcarbamylase deficiency: An under recognized cause of metabolic encephalopathy

OpenAIRE

Rush, Eric T; Hartmann, Julianne E; Skrabal, Jill C; Rizzo, William B

2014-01-01

Introduction: Ornithine transcarbamylase deficiency is the most common inherited disorder of the urea cycle, has a variable phenotype, and is caused by mutations in the OTC gene. We report three cases of ornithine transcarbamylase deficiency to illustrate the late-onset presentation of this disorder and provide strategies for diagnosis and treatment. The patients were maternal first cousins, presenting with hyperammonemia and obtundation. Urea cycle disorder was not initially suspected in the...

Ornithine carbamoyltransferase from Spinacea oleracea: purification and characterization

Czech Academy of Sciences Publication Activity Database

Bellocco, E.; Di Salvo, C.; Lagana, G.; Galtieri, A.; Ficarra, S.; Kotyk, Arnošt; Leuzzi, U.

2002-01-01

Roč. 45, č. 4 (2002), s. 533-538 ISSN 0006-3134 Institutional research plan: CEZ:AV0Z5011922 Keywords : ornithine carbomoyltransferase * Spinacea oleracea Subject RIV: BE - Theoretical Physics Impact factor: 0.583, year: 2002

Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

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Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

2010-05-01

Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching.

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

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Crittenden, Christopher M.; Parker, W. Ryan; Jenner, Zachary B.; Bruns, Kerry A.; Akin, Lucas D.; McGee, William M.; Ciccimaro, Eugene; Brodbelt, Jennifer S.

2016-05-01

A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

Ornithine aminotransferase deficiency: Diagnostic difficulties in neonatal presentation

NARCIS (Netherlands)

Cleary, M. A.; Dorland, L.; de Koning, T. J.; Poll-The, B. T.; Duran, M.; Mandell, R.; Shih, V. E.; Berger, R.; Olpin, S. E.; Besley, G. T. N.

2005-01-01

We describe two unrelated cases of ornithine aminotransferase (OAT) deficiency with rare neonatal presentation of hyperammonaemia. The diagnosis in the neonatal presentation of OAT deficiency is hampered as hyperornithinaemia is absent. Enzyme and mutation studies confirmed the diagnosis. OAT

Biological Research for Radiation Protection

International Nuclear Information System (INIS)

Kim, In Gyu; Kim, Kug Chan; Jung, Il Lae; Choi, Yong Ho; Kim, Jin Sik; Moon, Myung Sook; Byun, Hee Sun; Phyo, Ki Heon; Kim, Sung Keun

2005-04-01

The work scope of 'Biological Research for the Radiation Protection' had contained the research about ornithine decarboxylase and its controlling proteins, thioredoxin, peroxiredoxin, S-adenosymethionine decarboxylase, and glutamate decarboxylase 67KD effect on the cell death triggered ionizing radiation and H 2 O 2 (toxic agents). In this study, to elucidate the role of these proteins in the ionizing radiation (or H 2 O 2 )-induced apoptotic cell death, we utilized sensesed (or antisensed) cells, which overexpress (or down-regulate) RNAs associated with these proteins biosynthesis, and investigated the effects of these genes on the cytotoxicity caused by ionizing radiation and H 2 O 2 (or paraquat). We also investigated whether genisteine(or thiamine) may enhance the cytotoxic efficacy of tumor cells caused by ionizing radiation (may enhance the preventing effect radiation or paraquat-induced damage) because such compounds are able to potentiate the cell-killing or cell protecting effects. Based on the above result, we suggest that the express regulation of theses genes have potentially importance for sensitizing the efficiency of radiation therapy of cancer or for protecting the radiation-induced damage of normal cells

Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme.

Science.gov (United States)

Persson, K; Aslund, L; Grahn, B; Hanke, J; Heby, O

1998-01-01

All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC. PMID:9677309

N-ω-chloroacetyl-L-ornithine has in-vitro activity against cancer cell lines and in-vivo activity against ascitic and solid tumors.

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Vargas-Ramírez, Alba L; Medina-Enríquez, Miriam M; Cordero-Rodríguez, Neira I; Ruiz-Cuello, Tatiana; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José G; Alcántara-Farfán, Verónica; Rodríguez-Páez, Lorena

2016-07-01

N-ω-chloroacetyl-L-ornithine (NCAO) is an ornithine decarboxylase (ODC) inhibitor that is known to exert cytotoxic and antiproliferative effects on three neoplastic human cancer cell lines (HeLa, MCF-7, and HepG2). Here, we show that NCAO has antiproliferative activity in 13 cancer cell lines, of diverse tissue origin from human and mice, and in a mouse cancer model in vivo. All cell lines were sensitive to NCAO after 72 h of treatment (the EC50 ranged from 1 to 50.6 µmol/l). The Ca Ski cell line was the most sensitive (EC50=1.18±0.07 µmol/l) and MDA-MB-231 was the least sensitive (EC50=50.6±0.3 µmol/l). This ODC inhibitor showed selectivity for cancer cells, exerting almost no cytotoxic effect on the normal Vero cell line (EC50>1000 µmol/l). NCAO induced apoptosis and inhibited tumor cell migration in vitro. Furthermore, in vivo, this compound (at 50 and 100 mg/kg, daily intraperitoneal injection for 7 days) exerted potent antitumor activity against both solid and ascitic tumors in a mouse model using the myeloma (Ag8) cell line. At these same two doses, the toxicological evaluation showed that NCAO has no obvious systemic toxicity. The current results suggest that the antitumor activity is exerted by apoptosis related not only to a local but also a systemic cytotoxic effect exerted by NCAO on tumor cells. The applications for NCAO as an antitumor agent may be extensive; however, further studies are needed to ascertain the antitumor activity on other types of tumor in vivo and to determine the precise molecular mechanism of its activity.

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

International Nuclear Information System (INIS)

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 β-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- 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, β-sulfopyruvate, was studied, and it was found that L-[1- 14 C]cysteinesulfonate is an accurate and convenient probe for cysteinesulfinate decarboxylase activity. Using L-[1- 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

Uncovering the Lactobacillus plantarum WCFS1 Gallate Decarboxylase Involved in Tannin Degradation

Science.gov (United States)

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

Control of utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12.

OpenAIRE

Shaibe, E; Metzer, E; Halpern, Y S

1985-01-01

The regulation of the synthesis of the enzymes involved in the utilization of L-arginine, L-ornithine, agmatine, and putrescine as a sole nitrogen source in Escherichia coli K-12 was examined. The synthesis of agmatine ureohydrolase, putrescine aminotransferase, and pyrroline dehydrogenase is dually controlled by catabolite repression and nitrogen availability. Catabolite repression of agmatine ureohydrolase, but not that of putrescine aminotransferase or pyrroline dehydrogenase, is relieved ...

Combined Use of α‐Difluoromethylornithine and an Inhibitor of S‐Adenosylmethionine Decarboxylase in Mice Bearing P388 Leukemia or Lewis Lung Carcinoma

Science.gov (United States)

Nakaike, Shiro; Kashiwagi, Keiko; Terao, Kiyoshi; Iio, Kokoro

1988-01-01

The antitumor and antimetastatic effects of α‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, combined with an inhibitor of S‐adenosylmethionine decarboxylase, either methylglyoxal bis(guanylhydrazone) (MGBG) or ethylglyoxal bis(guanylhydrazone) (EGBG), were studied in mice bearing P388 leukemia or Lewis lung carcinoma. Although EGBG is a more specific inhibitor of polyamine biosynthesis than the widely used MGBG, the antitumor effect of the DFMO‐EGBG combination on P388 leukemia‐bearing mice was less than that of the DFMO‐MGBG combination. The prolongation of survival time by the DFMOC1000 mg/kg)‐MGBG(25 mg/kg) combination was 2.65‐fold, while that of the DFMO(1000 mg/kg)‐EGBG(50 mg/kg) combination was 1.34‐fold. When mice were fed a polyamine‐deficient diet, stronger antitumor effects were exerted; the prolongation of survival time by the DFMO‐MGBG and the DFMO‐EGBG combinations was 2.89‐fold and 2.03‐fold, respectively. The antitumor effect of combined use of the two polyamine antimetabolites with mice on normal and polyamine‐deficient diets correlated with a decrease of polyamine charge contents in the tumor cells. The above in vivo results were confirmed clearly in the KB cell culture system. The antimetastatic activity of DFMO on Lewis lung carcinoma‐bearing mice was strengthened by the addition of MGBG or EGBG. The antimetastatic activity of the DFMO‐MGBG or DFMO‐EGBG combination did not parallel the polyamine charge contents in the primary tumor and blood. PMID:3133338

Changes in the biochemical composition of testes during spermatogenesis in Asterias vulgaris, with emphasis on the role of polyamines in regulating proliferation

International Nuclear Information System (INIS)

Smith, F.F.

1985-01-01

Testes of Asterias vulgaris are potentially useful for investigating mechanisms regulating spermatogenic events because of their structural simplicity and annual spermatogenic cycle. Examination of major biochemical classes during the spermatogenic cycle provides a definition of the changing chemical microenvironment influencing germinal cells and also suggests temporal relationships among successive spermatogenic events. Testes from seastars collected throughout the year were homogenized and lyophilized and aliquots assayed for DNA, RNA, total protein, free amino acids, total lipids, glycogen, and other carbohydrates; spermatogenic stage was determined by examination of paraffin sections. Activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, increases during the proliferative phase of spermatogenesis. Testicular ODC activity correlates well with DNA synthetic rate. To test the possible role of polyamines in regulating initiation of spermatogonial mitoses, intact testes near the beginning of the proliferative phase were incubated in vitro with exogenous polyamines. They subsequently showed a significant increase in incorporation of 3 H-thymidine into DNA. These results suggest a direct role for polyamines in the regulation of spermatogenic proliferation in a. vulgaris. Evidence for a regulatory role of polyamines in the initiation of proliferation, together with existing information on the environmental, hormonal, and cytological interactions, facilitates development of a preliminary model for regulation and entrainment of spermatogenesis

Regulation of intestinal mucosal growth by amino acids.

Science.gov (United States)

Ray, Ramesh M; Johnson, Leonard R

2014-03-01

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the

The preparation of 3-aminoxy-1-amino[1,1'-3H2]propane

International Nuclear Information System (INIS)

Pankaskie, M.C.; Scholtz, S.J.

1989-01-01

3-Aminoxy-1-aminopropane (APA) has previously been shown to be a potent inhibitor of the polyamine biosynthesis enzymes ornithine decarboxylase, adenosylmethionine decarboxylase, and spermidine synthase. Little information is known, however, regarding its mechanism of action, binding site mode(s), or cellular distribution. This report presents a relatively simple three step synthesis of 3-aminoxy-1-amino[1,1'- 3 H 2 ]propane via the catalytic tritiation of 3-aminoxypropionitrile hydrochloride. (author)

Polyamine biosynthesis during germination of yeast ascospores.

Science.gov (United States)

Brawley, J V; Ferro, A J

1979-01-01

The role of the diamine putrescine during germination and outgrowth of ascospores of Saccharomyces cerevisiae was examined. Ornithine decarboxylase activity increased and declined rapidly during germination and outgrowth; peak activity was attained after the cells had proceeded through the G1 interval of the cell cycle, whereas minimal activity was present at the completion of the first cell division. alpha-Methylornithine inhibited both ornithine decarboxylase activity and the in vivo accumulation of putrescine. In the presence of alpha-methylornithireak dormancy and proceed through one cell division. Subsequent cellular growth, however, was retarded but not completely inhibited. The supplementation of Methylglyoxal bis(guanylhydrazone) to sporulation medium greatly inhibited this sexual process. These data suggest that the synthesis of putrescine is not required for the breaking of spore dormancy, but that polyamine biosynthesis may be essential for meiosis and sporulation. PMID:387744

Role of polyols in thermal inactivation of shark ornithine transcarbamoylase

Czech Academy of Sciences Publication Activity Database

Bellocco, E.; Lagana, G.; Barreca, D.; Ficarra, S.; Tellone, E.; Magazu, S.; Branca, C.; Kotyk, Arnošt; Galtieri, A.; Leuzzi, U.

2005-01-01

Roč. 54, č. 4 (2005), s. 395-402 ISSN 0862-8408 Institutional research plan: CEZ:AV0Z5011922 Keywords : ornithine transcarbamoylase * thermal inactivation * shark enzyme Subject RIV: CE - Biochemistry Impact factor: 1.806, year: 2005

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

Science.gov (United States)

Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

2016-07-01

We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells.

Science.gov (United States)

Seppänen, P; Fagerström, R; Alhonen-Hongisto, L; Elo, H; Lumme, P; Jänne, J

1984-07-15

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in

Methyl salicylate-induced arginine catabolism is associated with up-regulation of polyamine and nitric oxide levels and improves chilling tolerance in cherry tomato fruit.

Science.gov (United States)

Zhang, Xinhua; Shen, Lin; Li, Fujun; Meng, Demei; Sheng, Jiping

2011-09-14

The effects of methyl salicylate (MeSA) on chilling injury (CI) and gene expression levels, enzyme activities, and metabolites related to arginine catabolism in cherry tomato fruit were investigated. Freshly harvested fruits were treated with 0.05 mM MeSA vapor at 20 °C for 12 h and then stored at 2 °C for up to 28 days. MeSA reduced CI and enhanced the accumulation of putrescine, spermidine, and spermine, which was associated with increased gene expression levels and activities of arginase, arginine decarboxylase, and ornithine decarboxylase at most sampling times. MeSA also increased nitric oxide synthase activity, which at least partly contributed to the increased nitric oxide content. The results indicate that MeSA activates the different pathways of arginine catabolism in cold-stored fruit and that the reduction in CI by MeSA may be due to the coordinated metabolism of arginine and the increase in polyamines and nitric oxide levels.

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

Science.gov (United States)

Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

2014-04-01

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

Directory of Open Access Journals (Sweden)

Schneider Anja

2003-01-01

Full Text Available Abstract Background Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. Results High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2 of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. Conclusion AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.

Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

Science.gov (United States)

Wang, Meizhou; Xu, Meijuan; Rao, Zhiming; Yang, Taowei; Zhang, Xian

2015-11-01

L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.

Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

Science.gov (United States)

Cheetham, B F; Shaw, D C; Bellett, A J

1982-01-01

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation. PMID:7177112

Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators.

Science.gov (United States)

Hudec, Jozef; Burdová, Mária; Kobida, L'ubomír; Komora, Ladislav; Macho, Vendelín; Kogan, Grigorij; Turianica, Ivan; Kochanová, Radka; Lozek, Otto; Habán, Miroslav; Chlebo, Peter

2007-07-11

The changes of the antioxidant (AOA) and antiradical activities (ARA) and the total contents of phenolics, anthocyanins, flavonols, and hydroxybenzoic acid in roots and different aerial sections of Echinacea purpurea, nettle, and dandelion, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG) were analyzed spectrophotometrically; hydroxycinnamic acids content was analyzed by RP-HPLC with UV detection. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in all herb sections, with the exception of Echinacea stems after treatment with KF. In root tissues IP was dramatically elevated mainly after CCHG application: 8.5-fold in Echinacea, 4.14-fold in nettle, and 2.08-fold in dandelion. ARA decrease of Echinacea leaves treated with regulators was in direct relation only with cichoric acid and caftaric acid contents. Both regulators uphold the formation of cinnamic acid conjugates, the most expressive being that of cichoric acid after treatment with CCHG in Echinacea roots from 2.71 to 20.92 mg g(-1). There was a strong relationship between increase of the total phenolics in all sections of Echinacea, as well as in the studied sections of dandelion, and the anthocyanin content.

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

Science.gov (United States)

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.

Comparison of ultraviolet light-induced skin carcinogenesis and ornithine decarboxylase activity in sencar and hairless SKH-1 mice fed a constant level of dietary lipid varying in corn and coconut oil

International Nuclear Information System (INIS)

Berton, T.R.; Fischer, S.M.; Conti, C.J.; Locniskar, M.F.

1996-01-01

To investigate the effect of various levels of corn oil and coconut oil on ultraviolet (UV) light‐induced skin tumorigenesis and ornithine decarboxylase (ODC) activity, Sencar and SKH‐1 mice were fed one of three 15% (weight) fat semipurified diets containing three ratios of com oil to coconut oil: 1.0%:14.0%, 7.9%:7.1%, and 15.0%:0.0% in Diets A, B, and C, respectively. Groups of 30 Sencar and SKH‐1 mice were fed one of the diets for three weeks before UV irradiation; then both strains were UV irradiated with an initial dose of 90 mJ/cm2. The dose was given three times a week and increased 25% each week. For Sencar mice (irradiated 33 wks for a total dose of 48 J/cm2), tumor incidence reached a maximum of 60%, 60%, and 53% for Diets A, B, and C, respectively, with an overall average of one to two tumors per tumor‐bearing animal. For the SKH‐1 mice (irradiated 29 wks for a total dose of 18 J/cm2), all diet groups reached 100% incidence by 29 weeks, with approximately 12 tumors per tumor‐bearing mouse. No significant effect of dietary corn oil/coconut oil was found for tumor latency, incidence, or yield in either strain. The effect of increasing com oil on epidermal ODC activity in chronically UV‐irradiated Sencar and SKH‐1 mice was assessed Three groups of mice from each strain were fed one of the experimental diets and UV irradiated for six weeks. Sencar mice showed no increase in ODC activity until six weeks of treatment, when the levels of ODC activity in the UV‐irradiated mice fed Diet A were significantly higher than those in mice fed Diet B or Diet C: 1.27, 0.55, and 0.52 nmol/mg protein/hr, respectively. In the SKH‐1 mice, ODC activity was increased by the first week of UV treatment, and by three weeks of treatment a dietary effect was observed: ODC activity was significantly higher in mice fed Diet C (0.70 nmol/mg protein/hr) than in mice fed Diet A (0.18 nmol/mg protein/hr). Although there was no significant effect of dietary corn oil

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

Science.gov (United States)

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Testudinibacter aquarius gen. nov., sp nov., a member of the family Pasteurellaceae isolated from the oral cavity of freshwater turtles

DEFF Research Database (Denmark)

Hansen, Mie Johanne; Pennanen, Elin Anna Erica; Bojesen, Anders Miki

2016-01-01

Pasteurellaceae. However, they could be separated from existing genera of the Pasteurellaceae by the following test results: indole, ornithine decarboxylase and Voges-Proskauer positive; and methyl red, urease and PNPG (alpha-glucosidase) negative. No X- or V-factor requirement was observed. A zone of beta...

Systemic administration of insulin-like growth factor I (IGF-I) causes growth of the rat prostate

DEFF Research Database (Denmark)

Tørring, N; Vinter-Jensen, L; Pedersen, S B

1997-01-01

PURPOSE: To investigate the effects of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) on the rat prostate. In addition, we investigated the effect of ornithine decarboxylase (ODC) inhibition with alpha-diflouromethylornitine (DFMO) on the expected growth of the prostate.MA...

mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.

OpenAIRE

Kim, R Y; Gasser, R; Wistow, G J

1992-01-01

mu-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that mu-cryst...

Origin of the Putrescine-Producing Ability of the Coagulase-Negative Bacterium Staphylococcus epidermidis 2015B

NARCIS (Netherlands)

Coton, Emmanuel; Mulder, Niels; Coton, Monika; Pochet, Sylvie; Trip, Hein; Lolkema, Juke S.

A multiplex PCR method, aimed at the detection of genes associated with biogenic amine production, identified the odc gene encoding ornithine decarboxylase in 1 of 15 strains of Staphylococcus epidermidis. The ability of the positive strain, S. epidermidis 2015B, to produce putrescine in vitro was

Novel agmatine analogue, γ-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels

International Nuclear Information System (INIS)

Singh, Sushma; Jhingran, Anupam; Sharma, Ankur; Simonian, Alina R.; Soininen, Pasi; Vepsalainen, Jouko; Khomutov, Alex R.; Madhubala, Rentala

2008-01-01

The efficacy of γ-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K i value of ∼60 μM. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK a 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

Science.gov (United States)

He, Amanda; Penix, Stephanie R; Basting, Preston J; Griffith, Jessie M; Creamer, Kaitlin E; Camperchioli, Dominic; Clark, Michelle W; Gonzales, Alexandra S; Chávez Erazo, Jorge Sebastian; George, Nadja S; Bhagwat, Arvind A; Slonczewski, Joan L

2017-06-15

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS 5 insertion or IS-mediated deletion in cadC , while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR , yhiM , and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases ( hya and hyb ) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes ( dmsABC , frdABCD , hybABO , nikABCDE , and nrfAC ). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of

Biochemical properties and crystal structure of ethylmethylglyoxal bis(guanylhydrazone) sulfate--an extremely powerful novel inhibitor of adenosylmethionine decarboxylase.

Science.gov (United States)

Elo, H; Mutikainen, I; Alhonen-Hongisto, L; Laine, R; Jänne, J; Lumme, P

1986-01-01

Ethylmethylglyoxal bis(guanylhydrazone) (EMGBG) sulfate, an analog of the well-known anti-leukemic drug methylglyoxal bis(guanylhydrazone), was synthesized. It was shown to be an extremely powerful competitive inhibitor of eukaryotic S-adenosylmethionine decarboxylase, with an apparent Ki value 12 nM. Thus, it appears to be the most powerful known inhibitor of the enzyme, being almost an order of magnitude more powerful than the corresponding ethylglyoxal derivative. It neither inhibited the proliferation of mouse L1210 leukemia cells in vitro, nor did it potentiate the growth inhibition produced by alpha-difluoromethyl ornithine. In this respect, its properties are closely related to those of dimethylglyoxal, ethylglyoxal and propylglyoxal bis(guanylhydrazones), while in striking contrast to those of the antiproliferative glyoxal and methylglyoxal analogs. EMGBG also inhibited intestinal diamine oxidase activity (Ki 0.7 microM). EMGBG sulfate was crystallized from water, giving orthorhombic crystals (space group Pbcn). Their crystal and molecular structure was determined by X-ray diffraction methods. The carbon-nitrogen double bonds between the ethylmethylglyoxal part and the aminoguanidine moieties were found to have the same configuration as they are known to have in the salts of glyoxal, methylglyoxal and propylglyoxal bis(guanylhydrazones). The glyoxal bis(guanylhydrazone) chain of the EMGBG cation deviated strongly from planarity, thus differing dramatically from the corresponding chains of the glyoxal, methylglyoxal and propylglyoxal analogs.

Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions

Directory of Open Access Journals (Sweden)

Mario U Manto

2015-03-01

Full Text Available Autoantibodies to the smaller isoform of glutamate decarboxylase can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct glutamate decarboxylase autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal glutamate decarboxylase antibodies. We found that glutamate decarboxylase autoantibodies present in patients with stiff person syndrome (n = 7 and cerebellar ataxia (n = 15 recognized an epitope distinct from that recognized by glutamate decarboxylase autoantibodies present in patients with type 1 diabetes mellitus (n = 10 or limbic encephalitis (n = 4. We demonstrated that the administration of a monoclonal glutamate decarboxylase antibody representing this epitope specificity (1 disrupted in vitro the association of glutamate decarboxylase with γ-Aminobutyric acid containing synaptic vesicles, (2 depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect, (3 significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task, (4 markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm, and (5 induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of glutamate decarboxylase by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such glutamate decarboxylase antibodies could be envisioned.

Posterior fossa syndrome in a patient with an ornithine transcarbamylase deficiency

NARCIS (Netherlands)

Nedermeijer, S. C M; Van Den Hout, J.; Geleijns, C.; De Klerk, H.; Catsman-Berrevoets, C. E.

2015-01-01

The posterior fossa syndrome (PFS) is a well-known clinical entity and mainly occurs in children. Ornithine transcarbamylase deficiency (OTC) is the most common urea cycle disorder, which occurs in an estimated 1 per 50.000 live births in Japan. Symptoms are mostly due to hyperammonemia and include

Insights into the mutation-induced HHH syndrome from modeling human mitochondrial ornithine transporter-1.

Directory of Open Access Journals (Sweden)

Jing-Fang Wang

Full Text Available Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109-113. Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and

ORNITHINE TRANSCARBAMYLASE DEFICIENCY – THE REAL CAUSE OF “FAMILY CURSE”. A CASE REPORT

Directory of Open Access Journals (Sweden)

Zh. Sh. Bagomedova

2016-01-01

Full Text Available Ornithine transcarbamylase deficiency (type II hyperammonemia – X-linked metabolic disorder of the urea cycle, caused by mutations of the gene encoding ornithine transcarbamylase (OTC. Changes to the nervous system caused by degenerative processes in the gray and white matter of the cerebral hemispheres. The authors describe 1-year-old boy with ornithine transcarbamylase deficiency as a clinical example, with the onset of the disease in the first year of life, with refusal of food, vomiting, weakness and tiredness progressing to lethargy and unconsciousness, convulsive seizures, refusal from meat in the interictal period, delayed of psychomotor development. The child was admitted to the children’s intensive care unit in serious condition, unconscious with severe neurological symptoms. The clinical picture, the results of instrumental and laboratory examination and the presence of family history were the basis for the assumption of the hereditary origin of the disease. Genetic further examination was planned. In the context of children’s intensive care unit, the patient was undergoing of intensive therapy, which had no effect. Death occurred on the 5th day of hospitalization. To verify the diagnosis post-mortem autopsy was conducted, based on which was installed the immediate cause of death. In confirming the diagnosis is considered as tandem mass spectrometry, and DNA diagnostics.

Late-onset ornithine transcarbamylase deficiency: An under recognized cause of metabolic encephalopathy

Directory of Open Access Journals (Sweden)

Eric T Rush

2014-07-01

Full Text Available Introduction: Ornithine transcarbamylase deficiency is the most common inherited disorder of the urea cycle, has a variable phenotype, and is caused by mutations in the OTC gene. We report three cases of ornithine transcarbamylase deficiency to illustrate the late-onset presentation of this disorder and provide strategies for diagnosis and treatment. The patients were maternal first cousins, presenting with hyperammonemia and obtundation. Urea cycle disorder was not initially suspected in the first patient, delaying diagnosis. Results: Sequencing of the OTC gene showed a novel missense mutation, c.563G > C (p.G188A. Numerous family members were found to carry this mutation, which shows a trend toward later onset. Each urea cycle disorder has its own unique pattern of biochemical abnormalities, which differ from non-metabolic causes of critical illness. Conclusion: Regardless of age, clinical suspicion of a urea cycle disorder is important in encephalopathic patients to ensure quick diagnosis and definitive treatment of the underlying inborn error of metabolism.

Adaptative increase of ornithine production and decrease of ammonia metabolism in rat colonocytes after hyperproteic diet ingestion.

Science.gov (United States)

Mouillé, Béatrice; Robert, Véronique; Blachier, François

2004-08-01

Chronic high-protein consumption leads to increased concentrations of NH(4)(+)/NH(3) in the colon lumen. We asked whether this increase has consequences on colonic epithelial cell metabolism. Rats were fed isocaloric diets containing 20 (P20) or 58% (P58) casein as the protein source for 7 days. NH(4)(+)/NH(3) concentration in the colonic lumen and in the colonic vein blood as well as ammonia metabolism by isolated surface colonic epithelial cells was determined. After 2 days of consumption of the P58 diet, marked increases of luminal and colonic vein blood NH(4)(+)/NH(3) concentrations were recorded when compared with the values obtained in the P20 group. Colonocytes recovered from the P58 group were characterized at that time and thereafter by an increased capacity for l-ornithine and urea production through arginase (P diet consumption, however, the ammonia metabolism into l-citrulline was found lower (P < 0.01) when compared with the values measured in the colonocytes recovered from the P20 group despite any decrease in the related enzymatic activities (i.e., carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase). This decrease was found to coincide with a return of blood NH(4)(+)/NH(3) concentration in colonic portal blood to values close to the one recorded in the P20 group. In response to increased NH(4)(+)/NH(3) concentration in the colon, the increased capacity of the colonocytes to synthesize l-ornithine is likely to correspond to an elevated l-ornithine requirement for the elimination of excessive blood ammonia in the liver urea cycle. Moreover, in the presence of NH(4)Cl, colonocytes diminished their synthesis capacity of l-citrulline from l-ornithine, allowing a lower cellular utilization of this latter amino acid. These results are discussed in relationship with an adaptative process that would be related to both interorgan metabolism and to the role of the colonic epithelium as a first line of defense toward luminal NH(4)(+)/NH(3

Characterization of the tumor-promoting activity of m-chloroperoxybenzoic acid in SENCAR mouse skin and its inhibition by gallotannin, oligomeric proanthocyanidin, and their monomeric units

Science.gov (United States)

Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Fatima K. Johnson; Amy W. Davis; Steven W. Newell; Richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellett

1996-01-01

m-Chloroperoxybenzoic acid (CPBA). Which induces ornithine decarboxylase activity as much as 12-0- terradecanoyIp horbol-13-acetate (TPA ). was tested for its ability to induce DNA synthesis. bydroperoxide (HPx) production. and tumor promotion in mouse epidermis in vivo. After an early inhibition. CPBA stimulates DNA synthesis. A response which is maintained between 16...

Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

International Nuclear Information System (INIS)

Nemeti, Balazs; Gregus, Zoltan

2009-01-01

Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

Polyamines and meristematic activity in Zea mays roots

International Nuclear Information System (INIS)

Schwartz, M.; Arzee, T.; Cohen, Y.; Altman, A.

1989-01-01

Polyamine content and biosynthesis were determined in conjunction with meristematic activity and growth of Zea mays roots. Three types of developmental events were investigated: growth of intact primary roots, formation of lateral root primordia following main root decapitation, and activity of the quiescent center following root cap excision. A low ratio of putrescine/spermidine cotents was found to be salient feature of regions with high meristematic activity, in all 3 experimental systems. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities increased concomitantly with lateral root primordia development and activation of the quiescent center. An increase in the incorporation of arginine and ornithine into spermidine was found in meristematic zones. L-canavanine inhibited primary root elongation and formation of lateral primordia as well as ADC and ODC activity. Similar inhibitory effects were found with MGBG and CHA, both inhibitors of polyamine biosynthesis. A parallel study of ODC localization and DNA synthesis (using α- 14 C-DFMO and 3 H-thymidine microautoradiography, respectively) revealed than root zones with high meristematic activity are characterized by high ODC activity

Metabolic evolution and a comparative omics analysis of Corynebacterium glutamicum for putrescine production.

Science.gov (United States)

Li, Zhen; Shen, Yu-Ping; Jiang, Xuan-Long; Feng, Li-Shen; Liu, Jian-Zhong

2018-02-01

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.

Spectroscopic and thermodynamic properties of L-ornithine monohydrochloride

Energy Technology Data Exchange (ETDEWEB)

Raja, M. Dinesh [Department of Physics, Bharath University, Chennai – 600073 (India); Kumar, C. Maria Ashok; Arulmozhi, S.; Madhavan, J., E-mail: jmadhavang@yahoo.com [Department of Physics, Loyola College, Chennai – 600034 (India)

2015-06-24

L-Ornithine Monohydrochloride (LOMHCL) has been investigated with the help of B3LYP density functional theory with 6-31 G (d, p) basis set. Fourier transform infrared and Fourier transform Raman spectra is to identify the various functional groups. The theoretical frequencies showed very good agreement with experimental values. On the basis of the thermodynamic properties of the title compound at different temperatures have been calculated, revealing the correlations between standard heat capacities (C) standard entropies (S), and standard enthalpy changes (H) and temperatures. Second harmonic generation (SHG) efficiency of the grown crystal has been studied.

Keto-isovalerate decarboxylase enzymes and methods of use thereof

Science.gov (United States)

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.

Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

DEFF Research Database (Denmark)

Qin, Jiufu; Zhou, Yongjin J.; Krivoruchko, Anastasia

2015-01-01

intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor...

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.)

Science.gov (United States)

Slocum, R. D.; Richardson, D. P.

1991-01-01

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

Energy Technology Data Exchange (ETDEWEB)

Maes, Dominique, E-mail: dominique.maes@vub.ac.be; Crabeel, Marjolaine [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium); Van de Weerdt, Cécile; Martial, Joseph [Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée de la Chimie 3, B-4000 Liège (Belgium); Peeters, Eveline; Charlier, Daniël [Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels (Belgium); Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium)

2006-12-01

A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.

At the intersection of toxicology, psychiatry, and genetics: a diagnosis of ornithine transcarbamylase deficiency.

Science.gov (United States)

Sloas, Harold Andrew; Ence, Thomas C; Mendez, Donna R; Cruz, Andrea T

2013-09-01

Ornithine transcarbamylase (OTC) deficiency is a genetic disorder involving a mutation of the ornithine transcarbamylase gene, located on the short arm of the X chromosome (Xp21.1). This makes the expression of the gene most common in homozygous males, but heterozygous females can also be affected and may be more likely to suffer from serious morbidity. Most males present early in the neonatal period with more devastating outcomes than their female counterparts. Up to 34% will present in the first 30 days of life (J Pediatr 2001;138:S30). Females often have partially functioning mitochondria due to uneven distribution of the mutant gene secondary to lyonization (“X-chromosome Inactivation”. Genetics Home Reference, 2012). Occasionally, symptomatic females may not even present until they are placed under metabolic stress such as a severe illness, fasting, pregnancy, or new medication (Roth KS, Steiner RD. “Ornithine Transcarbamylase Deficiency”. EMedicine, 2012). The urea cycle is the body's primary tool for the disposal of excess nitrogen, which is generated by the routine metabolism of proteins and amino acids. Mitochondrial dysfunction impairs urea production and result in hyperammonemia (Semin Neonatol 2002;7:27). The sine qua non among all degrees of OTC deficiency at presentation is hyperammonemia. As in adults, children will have similar symptoms of encephalopathy, but this may be expressed differently depending on the child's developmental level. We present an unusual case of OTC deficiency in an older child with undifferentiated symptoms of an anticholinergic toxidrome, liver failure, iron overdose, and mushroom poisoning.

Ornithine: at the crossroads of multiple paths to amino acids and polyamines

Science.gov (United States)

Rajtilak Majumdar; Rakesh Minocha; Subhash. Minocha

2015-01-01

After the 20 amino acids that make up the proteins in all living organisms, ornithine perhaps occupies the most critical position among the non-protein amino acids. It sits at the crossroads of interconversions of glutamate and arginine on the one hand and the production of proline, polyamines and several alkaloids on the other: all products of tremendous importance to...

Perturbation of the Monomer-Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer

Energy Technology Data Exchange (ETDEWEB)

Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J. [IUPUI; (Purdue)

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 k_cat. 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.

A randomized, double-masked, placebo-controlled crossover trial on the effects of L-ornithine on salivary cortisol and feelings of fatigue of flushers the morning after alcohol consumption

Directory of Open Access Journals (Sweden)

Kokubo Takeshi

2013-02-01

Full Text Available Abstract Background Residual alcohol effects on physiological and psychological symptoms are commonly experienced the morning after alcohol consumption. The purpose of this study was to assess the effects of L-ornithine on subjective feelings and salivary stress markers the morning after alcohol consumption and to investigate whether L-ornithine acutely accelerates ethanol metabolism. Methods This study had a randomized, placebo-controlled, double-masked crossover design. Subjects were all healthy Japanese adults with the ‘flusher’ phenotype for alcohol tolerance. In experiment 1, 11 subjects drank 0.4 g/kg body weight alcohol 1.5 h before their usual bedtime. Half an hour after drinking, they ingested either a placebo or 400 mg ornithine. The next morning on awakening, subjects completed a questionnaire containing a visual analog scale (VAS, the Oguri-Shirakawa-Azumi sleep inventory MA version (OSA-MA, and a profile of mood states (POMS and collected a saliva sample for measurement of salivary stress markers (cortisol, secretory immunoglobulin A, and α-amylase. In experiment 2, placebo or 400 mg ornithine were administrated to 16 subjects both before and after drinking, and the feeling of drunkenness, breath ethanol concentration and one-leg standing time were repeatedly investigated until 180 min after alcohol consumption. Results There were significant decreases in “awareness”, “feeling of fatigue” and “lassitude” VAS scores and in “anger-hostility” and “confusion” POMS scores and a significant increase in “sleep length” in the OSA-MA test. Salivary cortisol concentrations on awakening were reduced after ornithine supplementation. There were no differences between ornithine and placebo in any of the subjective or physiological parameters of acute alcohol metabolism. Conclusions Taking 400 mg ornithine after alcohol consumption improved various negative feelings and decreased the salivary stress marker cortisol the

Effect of ethanol and low pH on citrulline and ornithine excretion and arc gene expression by strains of Lactobacillus brevis and Pediococcus pentosaceus.

Science.gov (United States)

Araque, Isabel; Bordons, Albert; Reguant, Cristina

2013-02-01

The accumulation of citrulline and ornithine in wine or beer as a result of the arginine catabolism of some lactic acid bacteria (LAB) species increases the risk of ethyl carbamate and putrescine formation, respectively. Several LAB species, which are found as spoilage bacteria in alcoholic beverages, have been reported to be arginine degrading. This study evaluates the effect of ethanol content and low pH on the excretion of citrulline and ornithine by two strains belonging to the potential contaminant species Lactobacillus brevis and Pediococcus pentosaceus. In the conditions that most affected cell viability, arginine consumption per cell increased noticeably, indicating that arginine utilization may be a stress responsive mechanism. L. brevis showed a higher accumulation of ornithine in the media than P. pentosaceus. In the presence of ethanol, a higher expression of the arcC gene was found in P. pentosaceus, which resulted in a lower excretion of citrulline and ornithine than in L. brevis. This suggests that L. brevis is more likely to produce these amino acids, which are precursors of ethyl carbamate and putrescine. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of Trypanosoma brucei brucei S-adenosyl-L-methionine decarboxylase and its inhibition by Berenil, pentamidine and methylglyoxal bis(guanylhydrazone).

Science.gov (United States)

Bitonti, A J; Dumont, J A; McCann, P P

1986-01-01

Trypanosoma brucei brucei S-adenosyl-L-methionine (AdoMet) decarboxylase was found to be relatively insensitive to activation by putrescine as compared with the mammalian enzyme, being stimulated by only 50% over a 10,000-fold range of putrescine concentrations. The enzyme was not stimulated by up to 10 mM-Mg2+. The Km for AdoMet was 30 microM, similar to that of other eukaryotic AdoMet decarboxylases. T.b. brucei AdoMet decarboxylase activity was apparently irreversibly inhibited in vitro by Berenil and reversibly by pentamidine and methylglyoxal bis(guanylhydrazone). Berenil also inhibited trypanosomal AdoMet decarboxylase by 70% within 4 h after administration to infected rats and markedly increased the concentration of putrescine in trypanosomes that were exposed to the drug in vivo. Spermidine and spermine blocked the curative effect of Berenil on model mouse T.b. brucei infections. This effect of the polyamines was probably not due to reversal of Berenil's inhibitory effects on the AdoMet decarboxylase. PMID:3800910

Stereospecific control of peptide gas-phase ion chemistry with cis and trans cyclo ornithine residues

Czech Academy of Sciences Publication Activity Database

Marek, Aleš; Nguyen, H. T. H.; Brož, Břetislav; Tureček, F.

2018-01-01

Roč. 53, č. 2 (2018), s. 124-137 ISSN 1076-5174 Institutional support: RVO:61388963 Keywords : cis and trans isomers * cyclo ornithine * peptide dissociations * peptide ion structures * stereochemistry Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 2.422, year: 2016

Structural Basis for Recognition of L-lysine, L-ornithine, and L-2,4-diamino Butyric Acid by Lysine Cyclodeaminase.

Science.gov (United States)

Min, Kyungjin; Yoon, Hye-Jin; Matsuura, Atsushi; Kim, Yong Hwan; Lee, Hyung Ho

2018-04-30

L-pipecolic acid is a non-protein amino acid commonly found in plants, animals, and microorganisms. It is a well-known precursor to numerous microbial secondary metabolites and pharmaceuticals, including anticancer agents, immunosuppressants, and several antibiotics. Lysine cyclodeaminase (LCD) catalyzes β-deamination of L-lysine into L-pipecolic acid using β-nicotinamide adenine dinucleotide as a cofactor. Expression of a human homolog of LCD, μ-crystallin, is elevated in prostate cancer patients. To understand the structural features and catalytic mechanisms of LCD, we determined the crystal structures of Streptomyces pristinaespiralis LCD (SpLCD) in (i) a binary complex with NAD + , (ii) a ternary complex with NAD + and L-pipecolic acid, (iii) a ternary complex with NAD + and L-proline, and (iv) a ternary complex with NAD + and L-2,4-diamino butyric acid. The overall structure of SpLCD was similar to that of ornithine cyclodeaminase from Pseudomonas putida . In addition, SpLCD recognized L-lysine, L-ornithine, and L-2,4-diamino butyric acid despite differences in the active site, including differences in hydrogen bonding by Asp236, which corresponds with Asp228 from Pseudomonas putida ornithine cyclodeaminase. The substrate binding pocket of SpLCD allowed substrates smaller than lysine to bind, thus enabling binding to ornithine and L-2,4-diamino butyric acid. Our structural and biochemical data facilitate a detailed understanding of substrate and product recognition, thus providing evidence for a reaction mechanism for SpLCD. The proposed mechanism is unusual in that NAD + is initially converted into NADH and then reverted back into NAD + at a late stage of the reaction.

Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii

Energy Technology Data Exchange (ETDEWEB)

Rivas, Blanca de las; Rodríguez, Héctor [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Angulo, Iván [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: xjosemi@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)

2007-07-01

The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His{sub 6} tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å{sup 3} Da{sup −1}, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model.

Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii

International Nuclear Information System (INIS)

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

2007-01-01

The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His 6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å 3 Da −1 , respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

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Marta Perez

2017-11-01

Full Text Available Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi was implicated in interaction among the two clusters.

Ornithine Decarboxylase G316A Genotype and Colorectal Cancer Risk

Czech Academy of Sciences Publication Activity Database

Hughes, D. J.; Hlavatá, I.; Souček, P.; Pardini, Barbara; Naccarati, Alessio; Vodičková, Ludmila; O´Morain, C. O.; Vodička, Pavel

2011-01-01

Roč. 13, č. 8 (2011), s. 860-864 ISSN 1462-8910 R&D Projects: GA ČR GA310/07/1430; GA ČR GP305/09/P194 Grant - others:GAUK(CZ) 15109/2009 Institutional research plan: CEZ:AV0Z50390512 Keywords : colorectal cancer * cancer genetics * association study Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.927, year: 2011

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity.

Science.gov (United States)

De Luca, Maria; Roshina, Nataliya V; Geiger-Thornsberry, Gretchen L; Lyman, Richard F; Pasyukova, Elena G; Mackay, Trudy F C

2003-08-01

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.

Moellerella wisconsensis, a new genus and species of Enterobacteriaceae found in human stool specimens.

OpenAIRE

Hickman-Brenner, F W; Huntley-Carter, G P; Saitoh, Y; Steigerwalt, A G; Farmer, J J; Brenner, D J

1984-01-01

The name Moellerella wisconsensis is proposed for a group of the family Enterobacteriaceae previously called enteric group 46. The species name, wisconsensis, was coined because six of the nine strains were isolated in Wisconsin. M. wisconsensis strains were negative for indole production, Voges-Proskauer, H2S production, urea, phenylalanine deaminase, lysine and ornithine decarboxylases, arginine dihydrolase, gas production from D-glucose, acid production from trehalose, and motility; the st...

Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

Science.gov (United States)

Baldin, Clara; Valiante, Vito; Krüger, Thomas; Schafferer, Lukas; Haas, Hubertus; Kniemeyer, Olaf; Brakhage, Axel A

2015-07-01

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Weissella halotolerans W22 combines arginine deiminase and ornithine decarboxylation pathways and converts arginine to putrescine

NARCIS (Netherlands)

Pereira, C. I.; San Romao, M. V.; Lolkema, J. S.; Barreto Crespo, M. T.; Baretto Crespo, M.

2009-01-01

Aims: To demonstrate that the meat food strain Weissella halotolerans combines an ornithine decarboxylation pathway and an arginine deiminase (ADI) pathway and is able to produce putrescine, a biogenic amine. Evidence is shown that these two pathways produce a proton motive force (PMF). Methods and

AUTOANTIBODIES TO GLUTAMIC ACID DECARBOXYLASE AS A PATHOGENETIC MARKER OF TYPE I DIABETES MELLITUS

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N. V. Piven

2011-01-01

Full Text Available Abstract. A new method of enzyme-linked immunosorbent assay (in solid-phase ELISA format has been developed to determine concentrations of autoantibodies to glutamic acid decarboxylase, as well as an evidencebased methodology is proposed for its medical implications, as a quantitative pathogenetic predictive marker of autoimmune diagnostics in type 1 diabetes mellitus. This technique could be implied for serial production of diagnostic reagent kits, aimed for detection of autoantibodies to glutamic acid decarboxylase by means of ELISA approach. (Med. Immunol., 2011, vol. 13, N 2-3, pp 257-260

Molecular Regulation of Histamine Synthesis

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Hua Huang

2018-06-01

Full Text Available Histamine is a critical mediator of IgE/mast cell-mediated anaphylaxis, a neurotransmitter and a regulator of gastric acid secretion. Histamine is a monoamine synthesized from the amino acid histidine through a reaction catalyzed by the enzyme histidine decarboxylase (HDC, which removes carboxyl group from histidine. Despite the importance of histamine, transcriptional regulation of HDC gene expression in mammals is still poorly understood. In this review, we focus on discussing advances in the understanding of molecular regulation of mammalian histamine synthesis.

l-Histidine Decarboxylase and Tourette's Syndrome

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Ercan-Sencicek, A. Gulhan; Stillman, Althea A.; Ghosh, Ananda K.; Bilguvar, Kaya; O'Roak, Brian J.; Mason, Christopher E.; Abbott, Thomas; Gupta, Abha; King, Robert A.; Pauls, David L.; Tischfield, Jay A.; Heiman, Gary A.; Singer, Harvey S.; Gilbert, Donald L.; Hoekstra, Pieter J.; Morgan, Thomas M.; Loring, Erin; Yasuno, Katsuhito; Fernandez, Thomas; Sanders, Stephan; Louvi, Angeliki; Cho, Judy H.; Mane, Shrikant; Colangelo, Christopher M.; Biederer, Thomas; Lifton, Richard P.; Gunel, Murat; State, Matthew W.

2010-01-01

Summary Tourette's syndrome is a common developmental neuropsychiatric disorder characterized by chronic motor and vocal tics. Despite a strong genetic contribution, inheritance is complex, and risk alleles have proven difficult to identify. Here, we describe an analysis of linkage in a two-generation pedigree leading to the identification of a rare functional mutation in the HDC gene encoding l-histidine decarboxylase, the rate-limiting enzyme in histamine biosynthesis. Our findings, together with previously published data from model systems, point to a role for histaminergic neurotransmission in the mechanism and modulation of Tourette's syndrome and tics. PMID:20445167

Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis

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Kendra Bufkin

2017-10-01

Full Text Available N-Hydroxylating monooxygenases (NMOs are involved in siderophore biosynthesis. Siderophores are high affinity iron chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by microorganisms and plants. Recently, a new siderophore named albachelin was isolated from a culture of Amycolatopsis alba growing under iron-limiting conditions. This work focuses on the expression, purification, and characterization of the NMO, abachelin monooxygenase (AMO from A. alba. This enzyme was purified and characterized in its holo (FAD-bound and apo (FAD-free forms. The apo-AMO could be reconstituted by addition of free FAD. The two forms of AMO hydroxylate ornithine, while lysine increases oxidase activity but is not hydroxylated and display low affinity for NADPH.

Gene expression profiles reveal key pathways and genes associated with neuropathic pain in patients with spinal cord injury.

Science.gov (United States)

He, Xijing; Fan, Liying; Wu, Zhongheng; He, Jiaxuan; Cheng, Bin

2017-04-01

Previous gene expression profiling studies of neuropathic pain (NP) following spinal cord injury (SCI) have predominantly been performed in animal models. The present study aimed to investigate gene alterations in patients with spinal cord injury and to further examine the mechanisms underlying NP following SCI. The GSE69901 gene expression profile was downloaded from the public Gene Expression Omnibus database. Samples of peripheral blood mononuclear cells (PBMCs) derived from 12 patients with intractable NP and 13 control patients without pain were analyzed to identify the differentially expressed genes (DEGs), followed by functional enrichment analysis and protein‑protein interaction (PPI) network construction. In addition, a transcriptional regulation network was constructed and functional gene clustering was performed. A total of 70 upregulated and 61 downregulated DEGs were identified in the PBMC samples from patients with NP. The upregulated and downregulated genes were significantly involved in different Gene Ontology terms and pathways, including focal adhesion, T cell receptor signaling pathway and mitochondrial function. Glycogen synthase kinase 3 β (GSK3B) was identified as a hub protein in the PPI network. In addition, ornithine decarboxylase 1 (ODC1) and ornithine aminotransferase (OAT) were regulated by additional transcription factors in the regulation network. GSK3B, OAT and ODC1 were significantly enriched in two functional gene clusters, the function of mitochondrial membrane and DNA binding. Focal adhesion and the T cell receptor signaling pathway may be significantly linked with NP, and GSK3B, OAT and ODC1 may be potential targets for the treatment of NP.

Urtica dioica Effect on Malonyl-CoA Decarboxylase

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Qujeq

2014-09-01

Full Text Available Background The malonyl-CoA decarboxylase (MCD, EC.4.1.1.9 enzyme regulates malonyl-CoA levels. The effect of aerial parts extracts of Urtica dioica (UD on MCD is poorly understood. Objectives The present experiment was undertaken to evaluate the effect of UD aerial parts extracts on MCD level. Materials and Methods In this experimental study, two groups of rats were used: normal and hyperglycemic group. Then UD aerial parts extracts (5 mg /500 µL administrated to the hyperglycemic group of rats and finally, the MCD and insulin levels were measured in both groups. Results Interestingly, we observed that the UD aerial parts extracts powder caused a significant (P < 0.05 increase in insulin level during the experiment, from the base level of 0.36 ± 0.07 μg/L to the peak value of 0.52 ± 0.15 μg/L. Also, it caused a significant (P < 0.05 decrease in MCD level, from the base level of 29.68 ±1.29 pg/mL to the bottom value of 22.12 ± 2.41 pg/mL. Conclusions The results of the present study indicate that UD aerial part extracts would decrease MCD level in hyperglycemic rats.

Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

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Tiina Rasila

Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

Science.gov (United States)

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

International Nuclear Information System (INIS)

Shanker, G.; Pieringer, R.A.

1986-01-01

Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125 [I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line

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Gescher Andreas

2003-01-01

Full Text Available Abstract Background Many tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines. Methods Cell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO2 from 14C-labelled substrate, and polyamine levels were measured by HPLC. Results I3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G2/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration. Conclusion While indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative

Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats

OpenAIRE

Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

2014-01-01

PURPOSE: To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. METHODS: One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fe...

EFFECT OF AERO-/ANAEROBIOSIS ON DECARBOXYLASE ACTIVITY OF SELECTED LACTIC ACID BACTERIA

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Stanislav Kráčmar

2010-05-01

Full Text Available Biogenic amines are undesirable compounds produced in foods mainly through bacterial decarboxylase activity. The aim of this study was to investigate some environmental conditions (particularly aero/anaerobiosis, sodium chloride concentration (0–2% w/w, and amount of lactose (0–1% w/w on the activity of tyrosine decarboxylase enzymes of selected six technological important Lactococcus lactis strains. The levels of parameters tested were chosen according to real situation in fermented dairy products technology (especially cheese-making. Tyramine was determined by the ion-exchange chromatography with post-column ninhydrine derivatization and spectrophotometric detection. Tyrosine decarboxylation occurred during the active growth phase. Under the model conditions used, oxygen availability had influence on tyramine production, anaerobiosis seemed to favour the enzyme activity because all L. lactis strains produced higher tyramine amount. doi:10.5219/43

Effect of hexoses on the levels of pyruvate decarboxylase in Mucor rouxii.

OpenAIRE

Barrera, C R; Corral, J

1980-01-01

Pyruvate decarboxylase activity in the dimorphic fungus Mucor rouxii increased 25- to 35-fold in yeastlike and mycelial cells grown in the presence of glucose as compared to the activity observed in mycelial cultures grown in the absence of glucose.

Glutamate decarboxylase activity in rat brain during experimental epileptic seizures induced by pilocarpine

Energy Technology Data Exchange (ETDEWEB)

Netopilova, M; Drsata, J [Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, 50005 Hradec Kralove (Czech Republic); Haugvicova, R; Kubova, H; Mares, P [Institute of Physiology, Czech Academy of Sciences, 14220 Prague (Czech Republic)

1998-07-01

Glutamate decarboxylase (GAD) activity was studied rat brain parts in a pilocarpine model of epileptic seizures. An increased enzyme activity was found in hippocampus a cerebellum during the acute phase of seizures, while the cortex and cerebellum showed increased GAD activity in the chronic phase of the process. Systematic administration of pilocarpine to rats induces status epilepticus. The aim of this research was to find out if seizures induced by pilocarpine are connected changes in glutamate decarboxylase activity, the enzyme that catalyzes synthesis of inhibitory neurotransmitter GABA. GAD was assayed by means of radiometric method using {sup 14}C-carboxyl-labelled glutamate and measurement of {sup 14}CO{sub 2} radioactivity. Obtained results suggest that pilocarpine seizures are connected with changes of GAD activity in individual parts of rat brain. (authors)

Glutamate decarboxylase activity in rat brain during experimental epileptic seizures induced by pilocarpine

International Nuclear Information System (INIS)

Netopilova, M.; Drsata, J.; Haugvicova, R.; Kubova, H.; Mares, P.

1998-01-01

Glutamate decarboxylase (GAD) activity was studied rat brain parts in a pilocarpine model of epileptic seizures. An increased enzyme activity was found in hippocampus a cerebellum during the acute phase of seizures, while the cortex and cerebellum showed increased GAD activity in the chronic phase of the process. Systematic administration of pilocarpine to rats induces status epilepticus. The aim of this research was to find out if seizures induced by pilocarpine are connected changes in glutamate decarboxylase activity, the enzyme that catalyzes synthesis of inhibitory neurotransmitter GABA. GAD was assayed by means of radiometric method using 14 C-carboxyl-labelled glutamate and measurement of 14 CO 2 radioactivity. Obtained results suggest that pilocarpine seizures are connected with changes of GAD activity in individual parts of rat brain. (authors)

Uroporphyrinogen decarboxylase is a radiosensitizing target for head and neck cancer.

Science.gov (United States)

Ito, Emma; Yue, Shijun; Moriyama, Eduardo H; Hui, Angela B; Kim, Inki; Shi, Wei; Alajez, Nehad M; Bhogal, Nirmal; Li, Guohua; Datti, Alessandro; Schimmer, Aaron D; Wilson, Brian C; Liu, Peter P; Durocher, Daniel; Neel, Benjamin G; O'Sullivan, Brian; Cummings, Bernard; Bristow, Rob; Wrana, Jeff; Liu, Fei-Fei

2011-01-26

Head and neck cancer (HNC) is the eighth most common malignancy worldwide, comprising a diverse group of cancers affecting the head and neck region. Despite advances in therapeutic options over the last few decades, treatment toxicities and overall clinical outcomes have remained disappointing, thereby underscoring a need to develop novel therapeutic approaches in HNC treatment. Uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified from an RNA interference-based high-throughput screen as a tumor-selective radiosensitizing target for HNC. UROD knockdown plus radiation induced caspase-mediated apoptosis and cell cycle arrest in HNC cells in vitro and suppressed the in vivo tumor-forming capacity of HNC cells, as well as delayed the growth of established tumor xenografts in mice. This radiosensitization appeared to be mediated by alterations in iron homeostasis and increased production of reactive oxygen species, resulting in enhanced tumor oxidative stress. Moreover, UROD was significantly overexpressed in HNC patient biopsies. Lower preradiation UROD mRNA expression correlated with improved disease-free survival, suggesting that UROD could potentially be used to predict radiation response. UROD down-regulation also radiosensitized several different models of human cancer, as well as sensitized tumors to chemotherapeutic agents, including 5-fluorouracil, cisplatin, and paclitaxel. Thus, our study has revealed UROD as a potent tumor-selective sensitizer for both radiation and chemotherapy, with potential relevance to many human malignancies.

The early history of polyamine research.

Science.gov (United States)

Bachrach, Uriel

2010-07-01

In 1678 Antonie van Leeuwenhoek identified crystalline substances in human semen. The structure of these crystals, named "spermine", was not elucidated by Rosenheim until 250 years later. Subsequently a triamine (spermidine) and a diamine (putrescine; 1,4-diaminobutane) were isolated from prokaryotic and eukaryotic systems. Soon it became apparent that polyamines can promote the growth of fastidious bacteria. Subsequently a group in Helsinki studied the accumulation of polyamines in regenerating rat liver, while Caldarera and his group studied polyamine synthesis in the developing chick embryo. These investigations led to metabolic studies. Ornithine decarboxylase was identified as a key enzyme in polyamine biosynthesis, while polyamine and diamine oxidations were studied by Mondovì. alpha-Diflouromethylornithine (DFMO) was synthesized by Merrell-Dow and became a potent inhibitor of ornithine decarboxylase. The findings of Russell that polyamines are excreted in the urine of cancer patients drew the attention of oncologists, who attempted the use new technologies for the detection of cancer and improving therapy. With the advance of molecular biology the structure of polyamine-biosynthetic enzymes was elaborated. Plants served as another important tool to study the physiological functions of polyamines. Bagni and his group at Bologna were pioneers in that field and for more than forty-six years set the foundation of a most interesting discipline. 2010 Elsevier Masson SAS. All rights reserved.

L-Arginine metabolism in cardiovascular and renal tissue from hyper- and hypothyroid rats.

Science.gov (United States)

Rodríguez-Gómez, Isabel; Moliz, Juan N; Quesada, Andrés; Montoro-Molina, Sebastian; Vargas-Tendero, Pablo; Osuna, Antonio; Wangensteen, Rosemary; Vargas, Félix

2016-03-01

This study assessed the effects of thyroid hormones on the enzymes involved in l-arginine metabolism and the metabolites generated by the different metabolic pathways. Compounds of l-arginine metabolism were measured in the kidney, heart, aorta, and liver of euthyroid, hyperthyroid, and hypothyroid rats after 6 weeks of treatment. Enzymes studied were NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]), arginases I and II, ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and l-arginine decarboxylase (ADC). Metabolites studied were l-arginine, l-citrulline, spermidine, spermine, and l-proline. Kidney heart and aorta levels of eNOS and iNOS were augmented and reduced (P hyperthyroid rats and was decreased in kidney and aorta of hypothyroid rats (P hyperthyroid rats and remained unchanged in all organs of hypothyroid rats. The substrate for these enzymes, l-arginine, was reduced (P hyperthyroid rats. Levels of ODC and spermidine, its product, were increased and decreased (P metabolic pathways. The changes recorded in the abundance of eNOS, arginases I and II, and ADC protein in renal and cardiovascular tissues may play a role in the hemodynamic and renal manifestations observed in thyroid disorders. Furthermore, the changes in ODC and spermidine might contribute to the changes in cardiac and renal mass observed in thyroid disorders. © 2015 by the Society for Experimental Biology and Medicine.

Enhancement of protocatechuate decarboxylase activity for the effective production of muconate from lignin-related aromatic compounds.

Science.gov (United States)

Sonoki, Tomonori; Morooka, Miyuki; Sakamoto, Kimitoshi; Otsuka, Yuichiro; Nakamura, Masaya; Jellison, Jody; Goodell, Barry

2014-12-20

The decarboxylation reaction of protocatechuate has been described as a bottleneck and a rate-limiting step in cis,cis-muconate (ccMA) bioproduction from renewable feedstocks such as sugar. Because sugars are already in high demand in the development of many bio-based products, our work focuses on improving protocatechuate decarboxylase (Pdc) activity and ccMA production in particular, from lignin-related aromatic compounds. We previously had transformed an Escherichia coli strain using aroY, which had been used as a protocatechuate decarboxylase encoding gene from Klebsiella pneumoniae subsp. pneumoniae A170-40, and inserted other required genes from Pseudomonas putida KT2440, to allow the production of ccMA from vanillin. This recombinant strain produced ccMA from vanillin, however the Pdc reaction step remained a bottleneck during incubation. In the current study, we identify a way to increase protocatechuate decarboxylase activity in E. coli through enzyme production involving both aroY and kpdB; the latter which encodes for the B subunit of 4-hydroxybenzoate decarboxylase. This permits expression of Pdc activity at a level approximately 14-fold greater than the strain with aroY only. The expression level of AroY increased, apparently as a function of the co-expression of AroY and KpdB. Our results also imply that ccMA may inhibit vanillate demethylation, a reaction step that is rate limiting for efficient ccMA production from lignin-related aromatic compounds, so even though ccMA production may be enhanced, other challenges to overcome vanilate demethylation inhibition still remain.

Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

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Manal Alkan

2015-01-01

Full Text Available Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD mouse model. To this end, we used mice (inactivated knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response.

Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes.

Science.gov (United States)

Kumar, Rahul

2016-01-01

Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

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

Science.gov (United States)

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)

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

Science.gov (United States)

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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

OpenAIRE

Alkan , Manal; Machavoine , François; Rignault , Rachel; Dam , Julie; Dy , Michel; Thieblemont , Nathalie

2015-01-01

International audience; Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC −/− m...

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

International Nuclear Information System (INIS)

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

2007-01-01

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 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 3 , with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å 3 Da −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

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

Energy Technology Data Exchange (ETDEWEB)

Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: xjosemi@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)

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.

Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats.

Science.gov (United States)

Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

2014-06-01

To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5 g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2-(ΔΔC)T method using the threshold cycle (CT) value for normalization. MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.

Effect of short-term ornithine alpha-ketoglutarate pretreatment on intestinal ischemia-reperfusion in rats

OpenAIRE

Gonçalves,Eduardo Silvio Gouveia; Rabelo,Camila Menezes; Prado Neto,Alberico Ximenes do; Garcia,José Huygens Parente; Guimarães,Sérgio Botelho; Vasconcelos,Paulo Roberto Leitão de

2011-01-01

PURPOSE: To investigate the effects of preventive enteral administration of ornithine alpha-ketoglutarate (OKG) in an ischemia-reperfusion rat model. METHODS: Sixty rats were randomized into five groups (G1-G5, n = 12). Each group was divided into two subgroups (n = 6) and treated with calcium carbonate (CaCa) or OKG by gavage. Thirty minutes later, the animals were anesthetized with xylazine 15mg + ketamine 1mg ip and subjected to laparotomy. G1-G3 rats served as controls. Rats in groups G4 ...

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

Science.gov (United States)

Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

2008-11-01

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

Identification of atypical Aeromonas salmonicida : Inter-laboratory evaluation and harmonization of methods

DEFF Research Database (Denmark)

Dalsgaard, Inger; Gudmundsdottir, B.K.; Helgason, S.

1998-01-01

the biochemical identification of atypical Aer. salmonicida before and after standardization of media and methods. Five laboratories examined 25 isolates of Aer. salmonicida from diverse fish species and geographical locations including the reference strains of Aer. salmonicida subsp, salmonicida (NCMB 1102......) and Aer. salmonicida subsp. achromogenes (NCMB 1110), Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase. The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical...

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1

Energy Technology Data Exchange (ETDEWEB)

Mydy, Lisa S.; Mashhadi, Zahra; Knight, T. William; Fenske, Tyler; Hagemann, Trevor; Hoppe, Robert W.; Han, Lanlan; Miller, Todd R.; Schwabacher, Alan W.; Silvaggi, Nicholas R. (UW); (Vanderbilt)

2017-11-14

The Gram-negative bacteriumSphingomonas wittichiiRW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, theS. wittichiigenome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure–function relationships within the ADCSF in general.

Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

Energy Technology Data Exchange (ETDEWEB)

Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh, E-mail: jvpratap@cdri.res.in

2015-01-09

Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

Regulation of ODC activity in the thymus and liver of rats by adrenal hormones.

Science.gov (United States)

Zahner, S L; Prahlad, K V; Mitchell, J L

1986-01-01

The activity of L-ornithine decarboxylase (EC 4.1.1.17, ODC) has become a useful indicator of hormone responsiveness. Various regimens of dexamethasone, aldosterone and epinephrine, alone or in combination, were administered to adrenalectomized rats either in acute or chronic doses. In addition, adrenalectomized rats, which were chronically treated with aldosterone and epinephrine, were given a single injection of 50 micrograms dexamethasone and sacrificed at various time intervals after hormone treatment. Hepatic and thymic ODC activity was measured. The expected dexamethasone effect, an increase in hepatic and a decrease in thymic ODC, was observed. This study also revealed that aldosterone induced similar responses in these tissues. Epinephrine had the opposite effect since chronic administration of dexamethasone or aldosterone with epinephrine resulted in control levels of ODC. Furthermore, when aldosterone and epinephrine were chronically administered to adrenalectomized rats, to study the acute effects of dexamethasone on rat thymus and liver, the time course of the response in each tissue was found to be distinct. The influence of the adrenal gland on rat thymus and liver is not restricted only to glucocorticoids, but may also involve other hormones which it secretes.

Benzoylformate analogues exhibit differential rate-determining steps in the benzoylformate decarboxylase reaction

International Nuclear Information System (INIS)

Garcia, G.A.; Weiss, P.M.; Cook, P.F.; Kenyon, G.L.; Cleland, W.W.

1987-01-01

Benzoylformate decarboxylase from Pseudomonas putida is a thiamine pyrophosphate (TPP)-dependent enzyme which converts benzoylformate to benzaldehyde and CO 2 . The rate-determining step(s) in the benzoylformate decarboxylase reaction for a series of substituted benzoylformates (p-CH 3 O, p-CH 3 , p-Cl, and m-F) were studied using solvent deuterium and 13 C kinetic isotope effects. The normal substrate was found to have two partially rate-determining steps; initial tetrahedral adduct formation (D 2 O-sensitive) and decarboxylation ( 13 C-sensitive). D 2 O and 13 C isotope effects indicate that electron-withdrawing substituents (p-Cl and m-F) remove the rate dependence upon decarboxylation such that only a D 2 O effect on (V/K) is observed. Conversely, electron-donating substituents increase the rate-dependence upon decarboxylation such that a larger 13 (V/K) is seen while the D 2 O effects on (V) and (V/K) are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate formed upon decarboxylation

Neurological disorders associated with glutamic acid decarboxylase antibodies: a Brazilian series

Directory of Open Access Journals (Sweden)

Maurício Fernandes

2012-09-01

Full Text Available Neurological disorders associated with glutamic acid decarboxylase (GAD antibodies are rare pleomorphic diseases of uncertain cause, of which stiff-person syndrome (SPS is the best-known. Here, we described nine consecutive cases of neurological disorders associated with anti-GAD, including nine patients with SPS and three cases with cerebellar ataxia. Additionally, four had hypothyroidism, three epilepsy, two diabetes mellitus and two axial myoclonus.

Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

DEFF Research Database (Denmark)

Zhang, Yiming; Liu, Guodong; Engqvist, Martin K. M.

2015-01-01

Background: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole...... DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase...... further increased the maximum specific growth rate to 0.069 h-1. Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing...

Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

Energy Technology Data Exchange (ETDEWEB)

Tomasi, Maria Lauda [Division of Gastrointestinal and Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); USC Research Center for Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); The Southern California Research Center for Alcoholic and Pancreatic Diseases and Cirrhosis, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); Ryoo, Minjung; Skay, Anna [Division of Gastrointestinal and Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); USC Research Center for Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); Tomasi, Ivan; Giordano, Pasquale [Department of Colorectal Surgery, Whipps Cross University Hospital, London E11 1NR (United Kingdom); Mato, José M. [CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Technology Park of Bizkaia, 48160 Derio, Bizkaia (Spain); Lu, Shelly C., E-mail: shellylu@usc.edu [Division of Gastrointestinal and Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); USC Research Center for Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States); The Southern California Research Center for Alcoholic and Pancreatic Diseases and Cirrhosis, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033 (United States)

2013-07-15

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100 pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell. -- Highlights: • MAT2A knockdown depletes putrescine and leads to apoptosis. • Putrescine attenuates MAT2A knockdown-induced apoptosis and growth

The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis

Directory of Open Access Journals (Sweden)

Marcus eFulde

2014-08-01

Full Text Available The arginine-ornithine antiporter (ArcD is part of the Arginine Deiminase System (ADS, a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-13C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth rate in chemically defined media supplemented with arginine when compared to the WT strain, indicating that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

Science.gov (United States)

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

Science.gov (United States)

Oliva, R; Vidal, S; Mezquita, C

1982-01-01

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration. Images PLATE 1 PMID:7159401

Structure-activity relationship study of sesquiterpene lactones and their semi-synthetic amino derivatives as potential antitrypanosomal products

CSIR Research Space (South Africa)

Zimmermann, S

2014-03-01

Full Text Available ,β-unsaturated nucleophilic enoate groups at C13 and C3' in 1 with trypanothione and glutathione in trypanosomes corroborated the long presumed mode of action of STLs, in addition to an inhibition of ornithine decarboxylase [8]. The same mode of action can be expected... control group of four mice was infected, but remained untreated. The determination of the parasitaemia was done on day 7 post infection. 6 μL of tail blood were diluted in 24 μL sodium citrate (3.2%), whereby the first μL was discarded to obtain...

Transcriptional response to deletion of the phosphatidylserine decarboxylase Psd1p in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Gsell, Martina; Mascher, Gerald; Schuiki, Irmgard; Ploier, Birgit; Hrastnik, Claudia; Daum, Günther

2013-01-01

In the yeast, Saccharomyces cerevisiae, the synthesis of the essential phospholipid phosphatidylethanolamine (PE) is accomplished by a network of reactions which comprises four different pathways. The enzyme contributing most to PE formation is the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) which catalyzes conversion of phosphatidylserine (PS) to PE. To study the genome wide effect of an unbalanced cellular and mitochondrial PE level and in particular the contribution of Psd1p to this depletion we performed a DNA microarray analysis with a ∆psd1 deletion mutant. This approach revealed that 54 yeast genes were significantly up-regulated in the absence of PSD1 compared to wild type. Surprisingly, marked down-regulation of genes was not observed. A number of different cellular processes in different subcellular compartments were affected in a ∆psd1 mutant. Deletion mutants bearing defects in all 54 candidate genes, respectively, were analyzed for their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2, ∆gph1 and ∆rsb1, were affected in one of these parameters. The possible link of these mutations to PE deficiency and PSD1 deletion is discussed.

Switch from Sodium Phenylbutyrate to Glycerol Phenylbutyrate Improved Metabolic Stability in an Adolescent with Ornithine Transcarbamylase Deficiency.

OpenAIRE

Laemmle Alexander; Stricker Tamar; Häberle Johannes

2016-01-01

A male patient, born in 1999, was diagnosed with ornithine transcarbamylase deficiency as neonate and was managed with a strict low-protein diet supplemented with essential amino acids, l-citrulline, and l-arginine as well as sodium benzoate. He had an extensive history of hospitalizations for hyperammonemic crises throughout childhood and early adolescence, which continued after the addition of sodium phenylbutyrate in 2009. In December 2013 he was switched to glycerol phenylbutyrate, and hi...

Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli

International Nuclear Information System (INIS)

Alexopoulos, Eftichia; Kanjee, Usheer; Snider, Jamie; Houry, Walid A.; Pai, Emil F.

2008-01-01

The structure of the decameric inducible lysine decarboxylase from E. coli was determined by SIRAS using a hexatantalum dodecabromide (Ta 6 Br 12 2+ ) derivative. Model building and refinement are under way. The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222 1 ; the Ta 6 Br 12 2+ cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta 6 Br 12 2+ -derivatized structure to 5 Å resolution. Many of the Ta 6 Br 12 2+ -binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006 ▶), J. Biol. Chem.281, 1532–1546

Association of the −243A>G, +61450C>A Polymorphisms of the Glutamate Decarboxylase 2 (GAD2) Gene with Obesity and Insu¬lin Level in North Indian Population

OpenAIRE

Jai PRAKASH; Balraj MITTAL; Shally AWASTHI; Neena SRIVASTAVA

2016-01-01

Background: Obesity associated with type 2 diabetes, and hypertension increased mortality and morbidity. Glutamate decarboxylase 2 (GAD2) gene is associated with obesity and it regulate food intake and insulin level. We investigated the association of GAD-2gene −243A>G (rs2236418) and +61450C>A (rs992990) polymorphisms with obesity and related phenotypes.Methods: Insulin, glucose and lipid levels were estimated using standard protocols. All subjects were genotyped (PCR-RFLP) method.Resu...

DPD epitope-specific glutamic acid decarboxylase GAD)65 autoantibodies in children with Type 1 diabetes

Science.gov (United States)

To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical dat...

Antileishmanial activity of berenil and methylglyoxal bis (guanylhydrazone) and its correlation with S-adenosylmethionine decarboxylase and polyamines.

Science.gov (United States)

Mukhopadhyay, R; Madhubala, R

1995-01-01

Leishmania donovani S-adenosyl-L-methionine (AdoMet) decarboxylase was found to show a growth related pattern. Methylglyoxal bis (guanylhydrazone) (MGBG) and Berenil inhibited the growth of Leishmania donovani promastigotes (strain UR6) in a dose dependent manner. The concentrations of MGBG and Berenil required for 50% inhibition of rate of growth were 67 and 47 microM, respectively. The growth inhibition of MGBG was partially reversed by spermidine (100 microM) and spermine (100 microM). Berenil inhibition of promastigote growth was partially reversed by 100 microM spermidine whereas 100 microM spermine did not result in any reversal of growth. The reduction in parasitemia in vitro by these inhibitors was accompanied by inhibition of AdoMet decarboxylase activity and spermidine levels.

Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

Energy Technology Data Exchange (ETDEWEB)

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.

Crystal structure and substrate specificity of Drosophila 3,4-dihydroxyphenylalanine decarboxylase.

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Qian Han

2010-01-01

Full Text Available 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.

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

Science.gov (United States)

Kallio, A; Jänne, J

1983-01-01

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone. PMID:6411077

On the influence of ionizing radiation on polyamine biosynthesis and content in animal cells and on the possibility of involvement of polyamines in the formation and recovery from radiation damage

International Nuclear Information System (INIS)

Rosiek, O.

1979-01-01

The initial section of this monograph provides a review of the present data on distribution, biosynthesis, catabolism and biological function of polyamines, putrescine, spermidine, and spermine in animal cells. The conclusion is drawn that there is a possibility of participation of these compounds in the formation and recovery from radiation damage. In the investigations presented in the experimental section, it was established that ionizing radiation can induce changes of the polyamine content and activity of the enzymes of polyamine metabolism (ornithine decarboxylase, S-adenosylmethionine decarboxylase, diamine oxidase, and polyamine oxidase) in animal cells. The results were also obtained which indicate that a close relationship exists between the post-irradiation synthesis and accumulation of polyamines and such recovery processes from radiation insult as restorative cell proliferation and repair of chromosome damage. Moreover, it was found that products of enzymatic and radiolytic oxidative deamination of spermidine and spermine can cause inhibition of cell proliferation and induction of chromosome aberrations. (author)

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

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Weber, Heike E; Gottardi, Manuela; Brückner, Christine; Oreb, Mislav; Boles, Eckhard; Tripp, Joanna

2017-05-15

Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate

Danish children born with glutamic acid decarboxylase-65 and islet antigen-2 autoantibodies at birth had an increased risk to develop type 1 diabetes

DEFF Research Database (Denmark)

Eising, Stefanie; Nilsson, Anita; Carstensen, Bendix

2011-01-01

A large, population-based case-control cohort was used to test the hypothesis that glutamic acid decarboxylase-65 (GAD65) and islet antigen-2 autoantibodies (IA-2A) at birth predict type 1 diabetes.......A large, population-based case-control cohort was used to test the hypothesis that glutamic acid decarboxylase-65 (GAD65) and islet antigen-2 autoantibodies (IA-2A) at birth predict type 1 diabetes....

Identification of the orotidine-5'-monophosphate decarboxylase gene of the oleaginous yeast Rhodosporidium toruloides.

Science.gov (United States)

Yang, Fan; Zhang, Sufang; Tang, Wei; Zhao, Zongbao K

2008-09-01

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer of great industrial potential, yet there is no effective genetic tool for rationally engineering this microorganism. To develop a marker recycling system, the orotidine-5'-monophosphate (OMP) decarboxylase gene of R. toruloides (RtURA3) was isolated using methods of degenerate polymerase chain reaction (PCR) together with rapid amplification of cDNA ends. The results showed that RtURA3 contains four extrons and three introns, and that the encoded polypeptide holds a sequence of 279 amino acid residues with significant homology to those of OMP decarboxylases from other yeasts. A shuttle vector pYES2/CT-RtURA3 was constructed via site-specific insertion of RtURA3 into the commercial vector pYES2/CT. Transformation of the shuttle vector into Saccharomyces cerevisiae BY4741, a URA3-deficient yeast strain, ensured the viability of the strain on synthetic dextrose agar plate without uracil, suggesting that the isolated RtURA3 was functionally equivalent to the URA3 gene from S. cerevisiae.

Maternal protein-energy malnutrition during early pregnancy in sheep impacts the fetal ornithine cycle to reduce fetal kidney microvascular development.

Science.gov (United States)

Dunford, Louise J; Sinclair, Kevin D; Kwong, Wing Y; Sturrock, Craig; Clifford, Bethan L; Giles, Tom C; Gardner, David S

2014-11-01

This paper identifies a common nutritional pathway relating maternal through to fetal protein-energy malnutrition (PEM) and compromised fetal kidney development. Thirty-one twin-bearing sheep were fed either a control (n=15) or low-protein diet (n=16, 17 vs. 8.7 g crude protein/MJ metabolizable energy) from d 0 to 65 gestation (term, ∼ 145 d). Effects on the maternal and fetal nutritional environment were characterized by sampling blood and amniotic fluid. Kidney development was characterized by histology, immunohistochemistry, vascular corrosion casts, and molecular biology. PEM had little measureable effect on maternal and fetal macronutrient balance (glucose, total protein, total amino acids, and lactate were unaffected) or on fetal growth. PEM decreased maternal and fetal urea concentration, which blunted fetal ornithine availability and affected fetal hepatic polyamine production. For the first time in a large animal model, we associated these nutritional effects with reduced micro- but not macrovascular development in the fetal kidney. Maternal PEM specifically impacts the fetal ornithine cycle, affecting cellular polyamine metabolism and microvascular development of the fetal kidney, effects that likely underpin programming of kidney development and function by a maternal low protein diet. © FASEB.

Acute metabolic decompensation due to influenza in a mouse model of ornithine transcarbamylase deficiency

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Peter J. McGuire

2014-02-01

Full Text Available The urea cycle functions to incorporate ammonia, generated by normal metabolism, into urea. Urea cycle disorders (UCDs are caused by loss of function in any of the enzymes responsible for ureagenesis, and are characterized by life-threatening episodes of acute metabolic decompensation with hyperammonemia (HA. A prospective analysis of interim HA events in a cohort of individuals with ornithine transcarbamylase (OTC deficiency, the most common UCD, revealed that intercurrent infection was the most common precipitant of acute HA and was associated with markers of increased morbidity when compared with other precipitants. To further understand these clinical observations, we developed a model system of metabolic decompensation with HA triggered by viral infection (PR8 influenza using spf-ash mice, a model of OTC deficiency. Both wild-type (WT and spf-ash mice displayed similar cytokine profiles and lung viral titers in response to PR8 influenza infection. During infection, spf-ash mice displayed an increase in liver transaminases, suggesting a hepatic sensitivity to the inflammatory response and an altered hepatic immune response. Despite having no visible pathological changes by histology, WT and spf-ash mice had reduced CPS1 and OTC enzyme activities, and, unlike WT, spf-ash mice failed to increase ureagenesis. Depression of urea cycle function was seen in liver amino acid analysis, with reductions seen in aspartate, ornithine and arginine during infection. In conclusion, we developed a model system of acute metabolic decompensation due to infection in a mouse model of a UCD. In addition, we have identified metabolic perturbations during infection in the spf-ash mice, including a reduction of urea cycle intermediates. This model of acute metabolic decompensation with HA due to infection in UCD serves as a platform for exploring biochemical perturbations and the efficacy of treatments, and could be adapted to explore acute decompensation in other

Inhibitors of polyamine metabolism: review article.

Science.gov (United States)

Wallace, H M; Fraser, A V

2004-07-01

The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

The DOPA decarboxylase (DDC) gene is associated with alerting attention.

Science.gov (United States)

Zhu, Bi; Chen, Chuansheng; Moyzis, Robert K; Dong, Qi; Chen, Chunhui; He, Qinghua; Li, Jin; Li, Jun; Lei, Xuemei; Lin, Chongde

2013-06-03

DOPA decarboxylase (DDC) is involved in the synthesis of dopamine, norepinephrine and serotonin. It has been suggested that genes involved in the dopamine, norepinephrine, and cholinergic systems play an essential role in the efficiency of human attention networks. Attention refers to the cognitive process of obtaining and maintaining the alert state, orienting to sensory events, and regulating the conflicts of thoughts and behavior. The present study tested seven single nucleotide polymorphisms (SNPs) within the DDC gene for association with attention, which was assessed by the Attention Network Test to detect three networks of attention, including alerting, orienting, and executive attention, in a healthy Han Chinese sample (N=451). Association analysis for individual SNPs indicated that four of the seven SNPs (rs3887825, rs7786398, rs10499695, and rs6969081) were significantly associated with alerting attention. Haplotype-based association analysis revealed that alerting was associated with the haplotype G-A-T for SNPs rs7786398-rs10499695-rs6969081. These associations remained significant after correcting for multiple testing by max(T) permutation. No association was found for orienting and executive attention. This study provides the first evidence for the involvement of the DDC gene in alerting attention. A better understanding of the genetic basis of distinct attention networks would allow us to develop more effective diagnosis, treatment, and prevention of deficient or underdeveloped alerting attention as well as its related prevalent neuropsychiatric disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Biochemical and ultrastructural changes in rabbit sclera after treatment with 7-methylxanthine, theobromine, acetazolamide, or L-ornithine

Science.gov (United States)

Trier, K.; Olsen, E. B.; Kobayashi, T.; Ribel-Madsen, S. M.

1999-01-01

AIMS—To examine a possible effect of 7-methylxanthine, theobromine, acetazolamide, or L-ornithine on the ultrastructure and biochemical composition of rabbit sclera.
METHODS—Groups of pigmented rabbits, six in each group, were dosed during 10 weeks with one of the substances under investigation, and one untreated group was the control. Samples of anterior and posterior sclera were taken for determination of hydroxyproline, hydroxylysine, proline, proteoglycans, uronic acids and dermatan sulphate, chondroitin sulphate, and hyaluronic acid. Sections were examined with electron microscopy, and the diameter of the individual collagen fibrils was measured.
RESULTS—Treatment with theobromine produced a significant increase in the contents of hydroxylysine, hydroxyproline, and proline in both anterior and posterior sclera, while 7-methylxanthine increased the contents of hydroxyproline and proline selectively in posterior sclera. Acetazolamide, on the other hand, significantly decreased the contents of hydroxyproline and proline in samples from anterior sclera. Uronic acids in both anterior and posterior sclera were significantly reduced by treatment with 7-methylxanthine, and L-ornithine significantly reduced uronic acids in posterior sclera. An inverse correlation between contents of hydroxyproline and uronic acids was found. The mean diameter of collagen fibrils was significantly higher in the posterior sclera from rabbits treated with 7-methylxanthine or theobromine, and significantly lower in rabbits treated with acetazolamide or L-ornithine compared with controls. In the anterior sclera, fibril diameter was significantly reduced in all treatment groups compared with controls. A positive, significant correlation between fibril diameter and content of hydroxyproline and proline was found in posterior sclera.
CONCLUSION—7-Methylxanthine, a metabolite of caffeine, increases collagen concentration and the diameter of collagen fibrils in the posterior

Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains

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Dorota Kręgiel

2009-01-01

Full Text Available Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1 is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

Energy Technology Data Exchange (ETDEWEB)

Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

1988-03-25

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human x-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, the authors introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.

Switch from Sodium Phenylbutyrate to Glycerol Phenylbutyrate Improved Metabolic Stability in an Adolescent with Ornithine Transcarbamylase Deficiency.

Science.gov (United States)

Laemmle, Alexander; Stricker, Tamar; Häberle, Johannes

2017-01-01

A male patient, born in 1999, was diagnosed with ornithine transcarbamylase deficiency as neonate and was managed with a strict low-protein diet supplemented with essential amino acids, L-citrulline, and L-arginine as well as sodium benzoate. He had an extensive history of hospitalizations for hyperammonemic crises throughout childhood and early adolescence, which continued after the addition of sodium phenylbutyrate in 2009. In December 2013 he was switched to glycerol phenylbutyrate, and his metabolic stability was greatly improved over the following 7 months prior to liver transplant.

Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

International Nuclear Information System (INIS)

Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C. K.

2010-01-01

The crystal structure of phenolic acid decarboxylase from B. pumilus strain UI-670 has been determined and refined at 1.69 Å resolution. The enzyme is a dimer, with each subunit adopting a β-barrel structure belonging to the lipocalin fold. The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site

A Molecular Dynamics (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) Study on Ornithine Cyclodeaminase (OCD): A Tale of Two Iminiums

Science.gov (United States)

Ion, Bogdan F.; Bushnell, Eric A. C.; De Luna, Phil; Gauld, James W.

2012-01-01

Ornithine cyclodeaminase (OCD) is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM) method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s Cα–H group to the NAD+ cofactor with concomitant formation of a Cα=NH2 + Schiff base with a barrier of 90.6 kJ mol−1. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the Cα=NH2 + intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the Cα-position. This is then followed by cleavage and loss of the α-NH2 group to give the Δ1-pyrroline-2-carboxylate that is subsequently reduced to L-proline. PMID:23202934

A Molecular Dynamics (MD and Quantum Mechanics/Molecular Mechanics (QM/MM Study on Ornithine Cyclodeaminase (OCD: A Tale of Two Iminiums

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James W. Gauld

2012-10-01

Full Text Available Ornithine cyclodeaminase (OCD is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD and a hybrid quantum mechanics/molecular mechanics (QM/MM method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s Cα–H group to the NAD+ cofactor with concomitant formation of a Cα=NH2+ Schiff base with a barrier of 90.6 kJ mol−1. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the Cα=NH2+ intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the Cα-position. This is then followed by cleavage and loss of the α-NH2 group to give the Δ1-pyrroline-2-carboxylate that is subsequently reduced to L-proline.

Limbic encephalitis with antibodies to glutamic acid decarboxylase presenting with brainstem symptoms

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Faruk Incecik

2015-01-01

Full Text Available Limbic encephalitis (LE is a neurological syndrome that may present in association with cancer, infection, or as an isolate clinical condition often accompanying autoimmune disorders. LE associated with glutamic acid decarboxylase antibodies (anti-GAD is rare in children. Here, we characterized the clinical and laboratory features of a patient presenting with brainstem involvement with non-paraneoplastic LE associated with anti-GAD antibodies. In our patient, after plasma exchange, we determined a dramatic improvement of the neurological deficits.

Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

Science.gov (United States)

Tavazzani, Elisa; Tritto, Simona; Spaiardi, Paolo; Botta, Laura; Manca, Marco; Prigioni, Ivo; Masetto, Sergio; Russo, Giancarlo

2014-01-01

The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

Cognitive decline in a patient with anti-glutamic acid decarboxylase autoimmunity; case report

OpenAIRE

Takagi, Masahito; Yamasaki, Hiroshi; Endo, Keiko; Yamada, Tetsuya; Kaneko, Keizo; Oka, Yoshitomo; Mori, Etsuro

2011-01-01

Abstract Background Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for producing γ-aminobutyric acid, and it has been suggested that antibodies against GAD play a role in neurological conditions and type 1 diabetes. However, it is not known whether dementia appears as the sole neurological manifestation associated with anti-GAD antibodies in the central nervous system. Case presentation We describe the clinical, neuropsychological, and neuroradiological findings of a 73-year-ol...

Biological research for the radiation protection

International Nuclear Information System (INIS)

Kim, In Gyu; Kim, Chan Kug; Shim, Hae Won; Jung, Il Lae; Byun, Hee Sun; Moon, Myung Sook; Cho, Hye Jeong; Kim, Jin Sik

2003-04-01

The work scope of 'Biological Research for the Radiation Protection' had contained the research about polyamine effect on cell death triggered ionizing radiation, H 2 O 2 and toxic agents. In this paper, to elucidate the role of polyamines as mediator in lysosomal damage and stress(H 2 O 2 )- induced apoptosis, we utilized α-DiFluoroMethylOrnithine (DFMO), which inhibited ornithine decarboxylase and depleted intracellular putrescine, and investigated the effects of polyamine on the apoptosis caused by H 2 O 2 , ionizing radiation and paraquat. We also showed that MGBG, inhibitor of polyamine biosynthesis, treatment affected intracellular redox steady states, intracellular ROS levels and protein oxidation. Thereafter we also investigated whether MGBG may enhance the cytotoxic efficacy of tumor cells caused by ionizing radiation or H 2 O 2 because such compounds are able to potentiate the cell-killing effects. In addition, ceruloplasmin and thioredoxin, possible antioxidant proteins, were shown to have protective effect on radiation- or H 2 O 2 (or chemicals)-induced macromolecular damage or cell death

Alpha-Difluoromethylornithine, an Irreversible Inhibitor of Polyamine Biosynthesis, as a Therapeutic Strategy against Hyperproliferative and Infectious Diseases

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Nicole LoGiudice

2018-02-01

Full Text Available The fluorinated ornithine analog α-difluoromethylornithine (DFMO, eflornithine, ornidyl is an irreversible suicide inhibitor of ornithine decarboxylase (ODC, the first and rate-limiting enzyme of polyamine biosynthesis. The ubiquitous and essential polyamines have many functions, but are primarily important for rapidly proliferating cells. Thus, ODC is potentially a drug target for any disease state where rapid growth is a key process leading to pathology. The compound was originally discovered as an anticancer drug, but its effectiveness was disappointing. However, DFMO was successfully developed to treat African sleeping sickness and is currently one of few clinically used drugs to combat this neglected tropical disease. The other Food and Drug Administration (FDA approved application for DFMO is as an active ingredient in the hair removal cream Vaniqa. In recent years, renewed interest in DFMO for hyperproliferative diseases has led to increased research and promising preclinical and clinical trials. This review explores the use of DFMO for the treatment of African sleeping sickness and hirsutism, as well as its potential as a chemopreventive and chemotherapeutic agent against colorectal cancer and neuroblastoma.

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

NARCIS (Netherlands)

Oud, B.; Flores, C.L.; Gancedo, C.; Zhang, X.; Trueheart, J.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

2012-01-01

Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards

The ArcD1 and ArcD2 arginine/ornithine exchangers encoded in the arginine deiminase (ADI) pathway gene cluster of Lactococcus lactis

NARCIS (Netherlands)

Noens, Elke E E; Kaczmarek, Michał B; Żygo, Monika; Lolkema, Juke S

2015-01-01

The arginine deiminase pathway (ADI) gene cluster in Lactococcus lactis contains two copies of a gene encoding an L-arginine/L-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion of arcD1 resulted in loss of

Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast

NARCIS (Netherlands)

A.J. van Maris; J.M. Geertman; A. Vermeulen; M.K. Groothuizen; A.A. Winkler; M.D. Piper; J.P. van Dijken; J.T. Pronk

2004-01-01

textabstractThe absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects:

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development.

Science.gov (United States)

Li, Wei-Dong; Huang, Min; Lü, Wen-Gang; Chen, Xiao; Shen, Ming-Hui; Li, Xiang-Min; Wang, Rong-Xia; Ke, Cai-Huan

2015-01-01

The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC) and antizyme (AZ) are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ) consisted of two overlapping open reading frames (ORFs) and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC) analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone.

Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit.

Science.gov (United States)

Yang, Xiaotang; Song, Jun; Du, Lina; Forney, Charles; Campbell-Palmer, Leslie; Fillmore, Sherry; Wismer, Paul; Zhang, Zhaoqi

2016-03-01

The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), β-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei.

Science.gov (United States)

Zimmermann, Stefanie; Oufir, Mouhssin; Leroux, Alejandro; Krauth-Siegel, R Luise; Becker, Katja; Kaiser, Marcel; Brun, Reto; Hamburger, Matthias; Adams, Michael

2013-11-15

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

AUTOANTIBODIES TO GLUTAMIC ACID DECARBOXYLASE AS A PATHOGENETIC MARKER OF TYPE I DIABETES MELLITUS

OpenAIRE

N. V. Piven; L. N. Lukhverchyk; A. I. Burakovsky; N. V. Polegenkaya; M. V. Karpovich

2011-01-01

Abstract. A new method of enzyme-linked immunosorbent assay (in solid-phase ELISA format) has been developed to determine concentrations of autoantibodies to glutamic acid decarboxylase, as well as an evidencebased methodology is proposed for its medical implications, as a quantitative pathogenetic predictive marker of autoimmune diagnostics in type 1 diabetes mellitus. This technique could be implied for serial production of diagnostic reagent kits, aimed for detection of autoantibodies to g...

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

Science.gov (United States)

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.

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

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De-Fen Shih

Full Text Available 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.

Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation.

Science.gov (United States)

Su, Marcia S; Schlicht, Sabine; Gänzle, Michael G

2011-08-30

Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri. A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ∆gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ∆gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ∆gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ∆gadB after 5 cycles of fermentation in back-slopped sourdough fermentations. The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.

Polyamine metabolism in ripening tomato fruit. II. Polyamine metabolism and synthesis in relation to enhanced putrescine content and storage life of alc tomato fruit

International Nuclear Information System (INIS)

Rastogi, R.; Davies, P.J.

1991-01-01

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele alc) and contain three times as much putrescine as the standard Rutgers variety (Alc) at the ripe stage. Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-alc-a near isogenic line possessing the allele alc, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in alc pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and alc fruit showed a decrease in the metabolism of [1,4- 14 C]putrescine and [terminal labeled- 3 H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-alc fruit, and as a result it was significantly higher in alc fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in alc fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Science.gov (United States)

Hopkins, D; Manchester, K L

1981-01-01

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed. PMID:7316998

A new mouse model of mild ornithine transcarbamylase deficiency (spf-j displays cerebral amino acid perturbations at baseline and upon systemic immune activation.

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Tatyana N Tarasenko

Full Text Available Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250 is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N. This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.

A novel gene signature for molecular diagnosis of human prostate cancer by RT-qPCR.

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Federica Rizzi

Full Text Available Prostate cancer (CaP is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC, ornithine decarboxylase antizyme (OAZ, adenosylmethionine decarboxylase (AdoMetDC, spermidine/spermine N(1-acetyltransferase (SSAT, histone H3 (H3, growth arrest specific gene (GAS1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH and Clusterin (CLU in tumour detection/classification of human CaP.The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP. No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold cross-validation procedure. CaP versus benign specimens were discriminated with (80+/-5% accuracy, (81+/-6% sensitivity and (78+/-7% specificity. The method also correctly classified 71% of patients with Gleason score or =7, an important predictor of final outcome.The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42% was considered CaP on the basis

The function of glycine decarboxylase complex is optimized to maintain high photorespiratory flux via buffering of its reaction products

DEFF Research Database (Denmark)

Bykova, Natalia V; Møller, Ian Max; Gardeström, Per

2014-01-01

oxidase. We discuss here possible mechanisms of high photorespiratory flux maintenance in mitochondria and suggest that it is fulfilled under conditions where the concentrations of glycine decarboxylase reaction products NADH and CO2 achieve an equilibrium provided by malate dehydrogenase and carbonic...

Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria

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Masome Zeinali

2016-09-01

Full Text Available Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours. Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples. Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable.

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

Energy Technology Data Exchange (ETDEWEB)

Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

2014-08-29

Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34{sup +}/K15{sup +}/p63{sup +} keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

International Nuclear Information System (INIS)

Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh; Elmets, Craig A.; Athar, Mohammad

2014-01-01

Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34 + /K15 + /p63 + keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in tumors induced

Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells

DEFF Research Database (Denmark)

Ramos-Molina, Bruno; López-Contreras, Andrés J; Lambertos, Ana

2015-01-01

Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane...... transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and V max of 17.......3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor...

The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

Science.gov (United States)

Hissen, Anna H T; Wan, Adrian N C; Warwas, Mark L; Pinto, Linda J; Moore, Margo M

2005-09-01

Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

Science.gov (United States)

S.C. Minocha; R. Minocha; A. Komamine

1991-01-01

Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

Science.gov (United States)

Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

2004-07-01

Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

Agdc1p - a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans.

Science.gov (United States)

Meier, Anna K; Worch, Sebastian; Böer, Erik; Hartmann, Anja; Mascher, Martin; Marzec, Marek; Scholz, Uwe; Riechen, Jan; Baronian, Kim; Schauer, Frieder; Bode, Rüdiger; Kunze, Gotthard

2017-01-01

Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (K m -0.7 ± 0.2 mM, k cat -42.0 ± 8.2 s -1 ) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (K m -3.2 ± 0.2 mM, k cat -44.0 ± 3.2 s -1 ). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δ agdc1 ] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis -muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be

Study of pyruvate decarboxylase and thiamine kinase from brewer's yeast by SERS

Science.gov (United States)

Maskevich, Sergei A.; Chernikevich, Ivan P.; Gachko, Gennedy A.; Kivach, Leonid N.; Strekal, Nataliya D.

1993-06-01

The Surface Enhanced Raman Scattering (SERS) spectra of holopyruvate decarboxylase (PDC) and thiamine kinase (ThK) adsorbed on silver electrode were obtained. In contrast to the Raman, the SERS spectrum of PDC contained no modes of tryptophan residues, it indicates a removal of this moiety from the surface. In the SERS spectrum of ThK the bands belonging to ligands bound to the protein were observed. A correlation between the SERS signal intensity and the enzymatic activity of the ThK separate fraction and found. The influence of amino acids on SERS spectra of thiamine (Th) was studied to determine the possible composition on microsurrounding of coenzyme.

Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase.

Science.gov (United States)

Pang, Jiayun; Scrutton, Nigel S; Sutcliffe, Michael J

2014-09-01

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multidisciplinary management of ornithine transcarbamylase (OTC) deficiency in pregnancy: essential to prevent hyperammonemic complications

Science.gov (United States)

Lamb, Stephanie; Aye, Christina Yi Ling; Murphy, Elaine; Mackillop, Lucy

2013-01-01

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error in the metabolism of the urea cycle with an incidence of 1 in 14 000 live births. Pregnancy can trigger potentially fatal hyperammonemic crises. We report a successful pregnancy in a 29-year-old primiparous patient with a known diagnosis of OTC deficiency since infancy. Hyperammonemic complications were avoided due to careful multidisciplinary management which included a detailed antenatal, intrapartum and postnatal plan. Management principles include avoidance of triggers, a low-protein diet and medications which promote the removal of nitrogen by alternative pathways. Triggers include metabolic stress such as febrile illness, particularly gastroenteritis, fasting and any protein loading. In our case the patient, in addition to a restricted protein intake, was prescribed sodium benzoate 4 g four times a day, sodium phenylbutyrate 2 g four times a day and arginine 500 mg four times a day to aid excretion of ammonia and reduce flux through the urea cycle. PMID:23283608

Ornithine Transcarbamylase Deficiency: If at First You Do Not Diagnose, Try and Try Again

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Christan D. Santos

2017-01-01

Full Text Available Ornithine transcarbamylase (OTC deficiency is well known for its diagnosis in the neonatal period. Presentation often occurs after protein feeding and manifests as poor oral intake, vomiting, lethargy progressing to seizure, respiratory difficulty, and eventually coma. Presentation at adulthood is rare (and likely underdiagnosed; however, OTC deficiency can be life-threatening and requires prompt investigation and treatment. Reports and guidelines are scarce due to its rarity. Here, we present a 59-year-old woman with a past history of irritable bowel syndrome who underwent a reparative operation for rectal prolapse and enterocele. Her postoperative course was complicated by a bowel perforation (which was repaired, prolonged mechanical ventilation, tracheostomy, critical illness myopathy, protein-caloric malnutrition, and altered mental status. After standard therapy for delirium failed, further investigation showed hyperammonemia and increased urine orotic acid, ultimately leading to the diagnosis of OTC deficiency. This case highlights the importance of considering OTC deficiency in hospitalized adults, especially during the diagnostic evaluation for altered mental status.

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula adeninivorans

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Anna K. Meier

2017-09-01

Full Text Available Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid, are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km −0.7 ± 0.2 mM, kcat −42.0 ± 8.2 s−1 than to protocatechuic acid (3,4-dihydroxybenzoic acid (Km −3.2 ± 0.2 mM, kcat −44.0 ± 3.2 s−1. Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans

Science.gov (United States)

Meier, Anna K.; Worch, Sebastian; Böer, Erik; Hartmann, Anja; Mascher, Martin; Marzec, Marek; Scholz, Uwe; Riechen, Jan; Baronian, Kim; Schauer, Frieder; Bode, Rüdiger; Kunze, Gotthard

2017-01-01

Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km −0.7 ± 0.2 mM, kcat −42.0 ± 8.2 s−1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km −3.2 ± 0.2 mM, kcat −44.0 ± 3.2 s−1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be

The Effect of Exogenous Spermidine Concentration on Polyamine Metabolism and Salt Tolerance in Zoysiagrass (Zoysia japonica Steud) Subjected to Short-Term Salinity Stress.

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Li, Shucheng; Jin, Han; Zhang, Qiang

2016-01-01

Salt stress, particularly short-term salt stress, is among the most serious abiotic factors limiting plant survival and growth in China. It has been established that exogenous spermidine (Spd) stimulates plant tolerance to salt stress. The present study utilized two zoysiagrass cultivars commonly grown in China that exhibit either sensitive (cv. Z081) or tolerant (cv. Z057) adaptation capacity to salt stress. The two cultivars were subjected to 200 mM salt stress and treated with different exogenous Spd concentrations for 8 days. Polyamine [diamine putrescine (Put), tetraamine spermine (Spm), and Spd], H2O2 and malondialdehyde (MDA) contents and polyamine metabolic (ADC, ODC, SAMDC, PAO, and DAO) and antioxidant (superoxide dismutase, catalase, and peroxidase) enzyme activities were measured. The results showed that salt stress induced increases in Spd and Spm contents and ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and diamine oxidase (DAO) activities in both cultivars. Exogenous Spd application did not alter polyamine contents via regulation of polyamine-degrading enzymes, and an increase in polyamine biosynthetic enzyme levels was observed during the experiment. Increasing the concentration of exogenous Spd resulted in a tendency of the Spd and Spm contents and ODC, SAMDC, DAO, and antioxidant enzyme activities to first increase and then decrease in both cultivars. H2O2 and MDA levels significantly decreased in both cultivars treated with Spd. Additionally, in both cultivars, positive correlations between polyamine biosynthetic enzymes (ADC, SAMDC), DAO, and antioxidant enzymes (SOD, POD, CAT), but negative correlations with H2O2 and MDA levels, and the Spd + Spm content were observed with an increase in the concentration of exogenous Spd.

Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

Science.gov (United States)

Jänne, J; Morris, D R

1984-03-15

Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development.

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Wei-Dong Li

Full Text Available The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC and antizyme (AZ are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ consisted of two overlapping open reading frames (ORFs and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone.

Current concepts on the physiology and genetics of neurotransmitters-mediating enzyme-aromatic L-amino acid decarboxylase

International Nuclear Information System (INIS)

Rahman, M.K.

1993-03-01

Two most important neurotransmitters, dopamine and serotonin are mediated by the enzyme aromatic L-amino acid decarboxylase (AADC). Because of their importance in the regulation of neuronal functions, behaviour and emotion of higher animals, many researchers are working on this enzyme to elucidate its physiological properties, structure and genetic aspects. We have discovered this enzyme in the mammalian blood, we established sensitive assay methods for the assay of the activities of this enzyme. We have made systematic studies on this enzyme in the tissues and brains of rats, and human subjects. We have found an endogenous inhibitor of this enzyme in the monkey's blood. The amino acid sequences of human AADC has been compared to rat or bovine. A full-length cDNA clone encoding human AADC has been isolated. Very recently the structure of human AADC gene including 5'-flaking region has been characterized and the transcriptional starting point has been determined. The human AADC gene assigned to chromosome 7. Up-to-date research data have shown that AADC is encoded by a single gene. Recently two patients with AADC deficiency were reported. This paper describes the systematic up-to-date review studies on AADC. (author). 62 refs, 5 figs, 8 tabs

Mechanisms of asbestos-induced squamous metaplasia in tracheobronchial epithelial cells

International Nuclear Information System (INIS)

Cameron, G.; Woodworth, C.D.; Edmondson, S.; Mossman, B.T.

1989-01-01

Within 1 to 4 weeks after exposure to asbestos, differentiated rodent and human tracheobronchial epithelial cells in organ culture undergo squamous metaplasia, a putative preneoplastic lesion characterized by conversion of mucociliary cell types to keratinizing cells. The exogenous addition of retinal acetate (RA) to culture medium of hamster tracheal organ cultures reverses preestablished, asbestos-induced squamous metaplasia, although data suggest that the effectiveness of RA decreases as the length of time between exposure to asbestos and initial application of RA increases. Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), inhibits squamous metaplasia caused by asbestos or vitamin A deficiency, whereas addition of methylglyoxal bis(guanyl-hydrazone) (MGBG), a structural analog of spermidine and inhibitor of S-adenosylmethionine decarboxylase, causes an enhancement of metaplasia under both circumstances. Basal cell hyperplasia and increased incorporation of 3 H-thymidine by tracheal epithelial cells also are seen after addition of the polyamines, putrescine or spermidine, to tracheal organ cultures, an observation supporting the importance of polyamines in the development of this lesion. The use of retinoids and inhibitors of ODC could be promising as preventive and/or therapeutic approaches for individuals at high risk for development of asbestos-associated diseases

Polyamines in plant physiology

Science.gov (United States)

Galston, A. W.; Sawhney, R. K.

1990-01-01

The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized. They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

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Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Angiotensin II-induced arterial thickening, fibrosis and stiffening involves elevated arginase function.

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Anil Bhatta

Full Text Available Arterial stiffness (AS is an independent risk factor for cardiovascular morbidity/mortality. Smooth muscle cell (SMC proliferation and increased collagen synthesis are key features in development of AS. Arginase (ARG, an enzyme implicated in many cardiovascular diseases, can compete with nitric oxide (NO synthase for their common substrate, L-arginine. Increased arginase can also provide ornithine for synthesis of polyamines via ornithine decarboxylase (ODC and proline/collagen via ornithine aminotransferase (OAT, leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced arterial thickening, fibrosis, and stiffness and that limiting its activity can prevent these changes.We tested this by studies in mice lacking one copy of the ARG1 gene that were treated with angiotensin II (Ang II, 4 weeks. Studies were also performed in rat aortic Ang II-treated SMC. In WT mice treated with Ang II, we observed aortic stiffening (pulse wave velocity and aortic and coronary fibrosis and thickening that were associated with increases in ARG1 and ODC expression/activity, proliferating cell nuclear antigen, hydroxyproline levels, and collagen 1 protein expression. ARG1 deletion prevented each of these alterations. Furthermore, exposure of SMC to Ang II (1 μM, 48 hrs increased ARG1 expression, ARG activity, ODC mRNA and activity, cell proliferation, collagen 1 protein expression and hydroxyproline content. Treatment with ABH prevented these changes.Arginase 1 is crucially involved in Ang II-induced SMC proliferation and arterial fibrosis and stiffness and represents a promising therapeutic target.

DOPA Decarboxylase Modulates Tau Toxicity.

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Kow, Rebecca L; Sikkema, Carl; Wheeler, Jeanna M; Wilkinson, Charles W; Kraemer, Brian C

2018-03-01

The microtubule-associated protein tau accumulates into toxic aggregates in multiple neurodegenerative diseases. We found previously that loss of D 2 -family dopamine receptors ameliorated tauopathy in multiple models including a Caenorhabditis elegans model of tauopathy. To better understand how loss of D 2 -family dopamine receptors can ameliorate tau toxicity, we screened a collection of C. elegans mutations in dopamine-related genes (n = 45) for changes in tau transgene-induced behavioral defects. These included many genes responsible for dopamine synthesis, metabolism, and signaling downstream of the D 2 receptors. We identified one dopamine synthesis gene, DOPA decarboxylase (DDC), as a suppressor of tau toxicity in tau transgenic worms. Loss of the C. elegans DDC gene, bas-1, ameliorated the behavioral deficits of tau transgenic worms, reduced phosphorylated and detergent-insoluble tau accumulation, and reduced tau-mediated neuron loss. Loss of function in other genes in the dopamine and serotonin synthesis pathways did not alter tau-induced toxicity; however, their function is required for the suppression of tau toxicity by bas-1. Additional loss of D 2 -family dopamine receptors did not synergize with bas-1 suppression of tauopathy phenotypes. Loss of the DDC bas-1 reduced tau-induced toxicity in a C. elegans model of tauopathy, while loss of no other dopamine or serotonin synthesis genes tested had this effect. Because loss of activity upstream of DDC could reduce suppression of tau by DDC, this suggests the possibility that loss of DDC suppresses tau via the combined accumulation of dopamine precursor levodopa and serotonin precursor 5-hydroxytryptophan. Published by Elsevier Inc.

Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

International Nuclear Information System (INIS)

Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

2006-01-01

Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å 3 Da −1 )

Structural basis of enzymatic activity for the ferulic acid decarboxylase (FADase from Enterobacter sp. Px6-4.

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Wen Gu

Full Text Available 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.

Assembly of an Oxalate Decarboxylase Produced under σK Control into the Bacillus subtilis Spore Coat

Science.gov (United States)

Costa, Teresa; Steil, Leif; Martins, Lígia O.; Völker, Uwe; Henriques, Adriano O.

2004-01-01

Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of σK and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme. PMID:14973022

Molecular cloning and expression of gene encoding aromatic amino acid decarboxylase in 'Vidal blanc' grape berries.

Science.gov (United States)

Pan, Qiu-Hong; Chen, Fang; Zhu, Bao-Qing; Ma, Li-Yan; Li, Li; Li, Jing-Ming

2012-04-01

The pleasantly fruity and floral 2-phenylethanol are a dominant aroma compound in post-ripening 'Vidal blanc' grapes. However, to date little has been reported about its synthetic pathway in grapevine. In the present study, a full-length cDNA of VvAADC (encoding aromatic amino acid decarboxylase) was firstly cloned from the berries of 'Vidal blanc', an interspecific hybrid variety of Vitis vinifera × Vitis riparia. This sequence encodes a complete open reading frame of 482 amino acids with a calculated molecular mass of 54 kDa and isoelectric point value (pI) of 5.73. The amino acid sequence deduced shared about 79% identity with that of aromatic L: -amino acid decarboxylases (AADCs) from tomato. Real-time PCR analysis indicated that VvAADC transcript abundance presented a small peak at 110 days after full bloom and then a continuous increase at the berry post-ripening stage, which was consistent with the accumulation of 2-phenylethanol, but did not correspond to the trends of two potential intermediates, phenethylamine and 2-phenylacetaldehyde. Furthermore, phenylalanine still exhibited a continuous increase even in post-ripening period. It is thus suggested that 2-phenylethanol biosynthetic pathway mediated by AADC exists in grape berries, but it has possibly little contribution to a considerable accumulation of 2-phenylethanol in post-ripening 'Vidal blanc' grapes.

Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

International Nuclear Information System (INIS)

Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia; Pegg, Anthony E.; Ealick, Steven E.

2008-01-01

The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction

Comprehensive clinical management of hirsutism.

Science.gov (United States)

Castelo-Branco, Camil; Cancelo, María Jesús

2010-07-01

Hirsutism is an excessive body and facial hair growth in women in locations where is normally minimal or absent following a hair-male pattern. For this reason is not uncommon that hirsutism raises psychological, cosmetic and social concerns. There are many treatment modalities that can be summarised into two broad groups: pharmacologic and non-pharmacologic treatment. Until now, medical treatment has been designed to interfere with the synthesis of androgen at the ovarian or adrenal level, or inhibit the effect of androgen at the receptor level, although recent progresses test other options such as insulin modulators or ornithine decarboxylase inhibitors. Mechanical treatment includes laser hair removal, electrolysis, depilatory creams, plucking and waxing. This article presents a general overview of hirsutism treatment options.

Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by β-catenin translocation and MMP7 activation

International Nuclear Information System (INIS)

Kimura, Ryota; Tanzawa, Hideki; Uzawa, Katsuhiro; Kasamatsu, Atsushi; Koyama, Tomoyoshi; Fukumoto, Chonji; Kouzu, Yukinao; Higo, Morihiro; Endo-Sakamoto, Yosuke; Ogawara, Katsunori; Shiiba, Masashi

2013-01-01

Glutamate decarboxylase 1 (GAD1), a rate-limiting enzyme in the production of γ-aminobutyric acid (GABA), is found in the GABAergic neurons of the central nervous system. Little is known about the relevance of GAD1 to oral squamous cell carcinoma (OSCC). We investigated the expression status of GAD1 and its functional mechanisms in OSCCs. We evaluated GAD1 mRNA and protein expressions in OSCC-derived cells using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. To assess the critical functions of GAD1, i.e., cellular proliferation, invasiveness, and migration, OSCC-derived cells were treated with the shRNA and specific GAD1 inhibitor, 3-mercaptopropionic acid (3-MPA). GAD1 expression in 80 patients with primary OSCCs was analyzed and compared to the clinicopathological behaviors of OSCC. qRT-PCR and immunoblotting analyses detected frequent up-regulation of GAD1 in OSCC-derived cells compared to human normal oral keratinocytes. Suppression of nuclear localization of β-catenin and MMP7 secretion was observed in GAD1 knockdown and 3-MPA-treated cells. We also found low cellular invasiveness and migratory abilities in GAD1 knockdown and 3-MPA-treated cells. In the clinical samples, GAD1 expression in the primary OSCCs was significantly (P < 0.05) higher than in normal counterparts and was correlated significantly (P < 0.05) with regional lymph node metastasis. Our data showed that up-regulation of GAD1 was a characteristic event in OSCCs and that GAD1 was correlated with cellular invasiveness and migration by regulating β-catenin translocation and MMP7 activation. GAD1 might play an important role in controlling tumoral invasiveness and metastasis in oral cancer

Improved Intranasal Retentivity and Transnasal Absorption Enhancement by PEGylated Poly-l-ornithine

Directory of Open Access Journals (Sweden)

Yusuke Kamiya

2018-01-01

Full Text Available We reported that the introduction of polyethylene glycol (PEG to poly-l-ornithine (PLO, which is an homopolymeric basic amino acid having absorption-enhancement ability, prolonged retention time in an in vitro inclined plate test, probably due to an increase in viscosity caused by PEGylation. The aim of the present study is to investigate whether the introduction of PEG chains to PLO improves intranasal retention and transnasal absorption in vivo. We performed intranasal administration experiments using PLO and PEG-PLO with a model drug, fluorescein isothiocyanate dextran (FD-4, in rats under closed and open systems. In the open system, transition of plasma FD-4 concentration after co-administration with unmodified PLO was low, and the area under the plasma concentration-time curve (AUC decreased to about 60% of that in the closed system. In contrast, the AUC after co-administration with PEG-PLO in the open system was about 90% of that in the closed system, and the transition of plasma FD-4 concentration and FD-4 absorption profile were similar to those of the closed system. These findings indicate that introducing PEG chains to homopolymeric basic amino acids (HPBAAs is a very useful method for developing a functional absorption enhancer that can exhibit an efficient in vivo absorption-enhancing effect.

OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese.

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Bo Kang

Full Text Available An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1, which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05. The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05. The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05, whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05. The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05. Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

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Kenneth W Yip

Full Text Available Uroporphyrinogen decarboxylase (UROD catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16, was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM, but did not affect porphobilinogen deaminase (at 62.5 µM, thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1. This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

Albizia lebbeck suppresses histamine signaling by the inhibition of histamine H1 receptor and histidine decarboxylase gene transcriptions.

Science.gov (United States)

Nurul, Islam Mohammed; Mizuguchi, Hiroyuki; Shahriar, Masum; Venkatesh, Pichairajan; Maeyama, Kazutaka; Mukherjee, Pulok K; Hattori, Masashi; Choudhuri, Mohamed Sahabuddin Kabir; Takeda, Noriaki; Fukui, Hiroyuki

2011-11-01

Histamine plays major roles in allergic diseases and its action is mediated mainly by histamine H(1) receptor (H1R). We have demonstrated that histamine signaling-related H1R and histidine decarboxylase (HDC) genes are allergic diseases sensitive genes and their expression level affects severity of the allergic symptoms. Therefore, compounds that suppress histamine signaling should be promising candidates as anti-allergic drugs. Here, we investigated the effect of the extract from the bark of Albizia lebbeck (AL), one of the ingredients of Ayruvedic medicines, on H1R and HDC gene expression using toluene-2,4-diisocyanate (TDI) sensitized allergy model rats and HeLa cells expressing endogenous H1R. Administration of the AL extract significantly decreased the numbers of sneezing and nasal rubbing. Pretreatment with the AL extract suppressed TDI-induced H1R and HDC mRNA elevations as well as [(3)H]mepyramine binding, HDC activity, and histamine content in the nasal mucosa. AL extract also suppressed TDI-induced up-regulation of IL-4, IL-5, and IL-13 mRNA. In HeLa cells, AL extract suppressed phorbol-12-myristate-13-acetate- or histamine-induced up-regulation of H1R mRNA. Our data suggest that AL alleviated nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through suppression of H1R and HDC gene transcriptions. Suppression of Th2-cytokine signaling by AL also suggests that it could affect the histamine-cytokine network. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2014-01-01

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 Ca2+-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 Ca2+ 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 Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-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. PMID:24799560

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

Science.gov (United States)

Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

2006-01-01

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

Characterization of molecular associations involving L-ornithine and α-ketoglutaric acid: crystal structure of L-ornithinium α-ketoglutarate.

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Allouchi, H; Céolin, R; Berthon, L; Tombret, F; Rietveld, I B

2014-07-01

The crystal structure of L-ornithinium α-ketoglutarate (C5H13N2O2, C5H5O5) has been solved by direct methods using single crystal X-ray diffraction data. It crystallizes in the monoclinic system, space group P21, unit cell parameters a=15.4326(3), b=5.2015(1), c=16.2067(3) Å and β=91.986(1)°, containing two independent pairs of molecular ions in the asymmetric unit. An extensive hydrogen-bond network and electrostatic charges due to proton transfer provide an important part of the cohesive energy of the crystal. The conformational versatility of L-ornithine and α-ketoglutaric acid is illustrated by the present results and crystal structures available from the Cambridge Structural Database. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity.

OpenAIRE

Leoni, L; Ciervo, A; Orsi, N; Visca, P

1996-01-01

The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site. RNA analysis showed that expression of PvdA is iron regulat...

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

OpenAIRE

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.

Molecular identification and characterization of the pyruvate decarboxylase gene family associated with latex regeneration and stress response in rubber tree.

Science.gov (United States)

Long, Xiangyu; He, Bin; Wang, Chuang; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

2015-02-01

In plants, ethanolic fermentation occurs not only under anaerobic conditions but also under aerobic conditions, and involves carbohydrate and energy metabolism. Pyruvate decarboxylase (PDC) is the first and the key enzyme of ethanolic fermentation, which branches off the main glycolytic pathway at pyruvate. Here, four PDC genes were isolated and identified in a rubber tree, and the protein sequences they encode are very similar. The expression patterns of HbPDC4 correlated well with tapping-simulated rubber productivity in virgin rubber trees, indicating it plays an important role in regulating glycometabolism during latex regeneration. HbPDC1, HbPDC2 and HbPDC3 had striking expressional responses in leaves and bark to drought, low temperature and high temperature stresses, indicating that the HbPDC genes are involve in self-protection and defense in response to various abiotic and biotic stresses during rubber tree growth and development. To understand ethanolic fermentation in rubber trees, it will be necessary to perform an in-depth study of the regulatory pathways controlling the HbPDCs in the future. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Relationship between 2-phenylethanol content and differential expression of L-amino acid decarboxylases (AADC) in (Vitis vinifera) vidal wine grape at different loads

International Nuclear Information System (INIS)

Lin, Y.; Li, K.; Liu, Z.; Guo, X.; Jiang, C.; Yue, G.; Li, W.; Dou, Y.; Zheng, J.

2018-01-01

In this study, the headspace-solid phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) was used to determine the type and content of aroma in Vidal grapes. A quantitative fluorescence measurement was performed to determine the differential expression of Amino Acid Decarboxylase (AADC). By conducting five different load treatments (fruit weight per 667 m2: 750, 1,000, 1,250, 1,500, and over 1,750 kg), we found that the main components of Vidal grapes were alcohols, esters, alkanes, aldehydes, phenols, ketones, and ethers. The relative levels of alcohols, esters, alkanes, and phenols were 25, 27, 18, and 14%, respectively. The relationship between the dynamic content of the characteristic aroma component 2-phenylethanol and the expression of AADC enzyme was explored. The results showed that for a small load, the relative expression levels of 2-phenylethanol-regulating AADC enzyme were high and low in the early and late stages of growth, respectively. For a large load, the content of 2-phenylethanol was low, while the relative expression levels of 2-phenylethanol-regulating AADC enzyme were low and high in the early and late stages of growth, respectively. In the early stage, the positive regulation was significant, and in the late stage, the relative expression of AADC was increased rapidly, which in turn, increased the positive regulation. It was recommended that the suitable yield for Vidal grape during peak fruiting period was 1,000~1,500 kg per 667 m2. This study provides the scientific basis for the control of fruit aroma and can be used as a reference for load adjustment in the production of wine grape during peak fruiting period. (author)

Haplotype analysis indicates an association between the DOPA decarboxylase (DDC) gene and nicotine dependence.

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Ma, Jennie Z; Beuten, Joke; Payne, Thomas J; Dupont, Randolph T; Elston, Robert C; Li, Ming D

2005-06-15

DOPA decarboxylase (DDC; also known as L-amino acid decarboxylase; AADC) is involved in the synthesis of dopamine, norepinephrine and serotonin. Because the mesolimbic dopaminergic system is implicated in the reinforcing effects of many drugs, including nicotine, the DDC gene is considered a plausible candidate for involvement in the development of vulnerability to nicotine dependence (ND). Further, this gene is located within the 7p11 region that showed a 'suggestive linkage' to ND in our previous genome-wide scan in the Framingham Heart Study population. In the present study, we tested eight single nucleotide polymorphisms (SNPs) within DDC for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerstrom test for ND (FTND) score, in a total of 2037 smokers and non-smokers from 602 nuclear families of African- or European-American (AA or EA, respectively) ancestry. Association analysis for individual SNPs using the PBAT-GEE program indicated that SNP rs921451 was significantly associated with two of the three adjusted ND measures in the EA sample (P=0.01-0.04). Haplotype-based association analysis revealed a protective T-G-T-G haplotype for rs921451-rs3735273-rs1451371-rs2060762 in the AA sample, which was significantly associated with all three adjusted ND measures after correction for multiple testing (min Z=-2.78, P=0.006 for HSI). In contrast, we found a high-risk T-G-T-G haplotype for a different SNP combination in the EA sample, rs921451-rs3735273-rs1451371-rs3757472, which showed a significant association after Bonferroni correction with the SQ and FTND score (max Z=2.73, P=0.005 for FTND). In summary, our findings provide the first evidence for the involvement of DDC in the susceptibility to ND and, further, reveal the racial specificity of its impact.

Diagnostic accuracy of the anti-glutamic acid decarboxylase antibody in type 1 diabetes mellitus: Comparison between radioimmunoassay and enzyme-linked immunosorbent assay.

Science.gov (United States)

Murata, Takashi; Tsuzaki, Kokoro; Nirengi, Shinsuke; Watanabe, Tomokazu; Mizutani, Yukako; Okada, Hayami; Tsukamoto, Masami; Odori, Shinji; Nakagawachi, Reiko; Kawaguchi, Yaeko; Yoshioka, Fumi; Yamada, Kazunori; Shimatsu, Akira; Kotani, Kazuhiko; Satoh-Asahara, Noriko; Sakane, Naoki

2017-07-01

The distributer of the anti-glutamic acid decarboxylase antibody assay kit using radioimmunoassay (RIA) recently announced its discontinuation, and proposed an alternative kit using enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to investigate the diagnostic values of the anti-glutamic acid decarboxylase antibody by RIA and ELISA among type 1 diabetes mellitus patients and control participants. A total of 79 type 1 diabetes mellitus patients and 79 age-matched controls were enrolled and assessed using RIA and ELISA. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for cut-off values (RIA = 1.5 U/mL and ELISA = 5.0 U/mL, respectively). Kappa coefficients were used to test for agreements between the RIA and ELISA methods regarding the diagnosis of type 1 diabetes mellitus. The sensitivity, specificity, positive predictive values, and negative predictive values for diagnosing type 1 diabetes mellitus were 57.0, 97.5, 95.7, and 69.4% by RIA, and 60.8, 100.0, 100.0 and 71.8% by ELISA, respectively. The diagnosis of type 1 diabetes mellitus using the RIA and ELISA methods showed substantial agreement with the kappa values of 0.74 for all participants, and of 0.64 for the acute type; however, there was moderate agreement with the kappa value of 0.56 for the slowly progressive type. The present study suggests that both anti-glutamic acid decarboxylase antibody by RIA and ELISA was useful for diagnosing type 1 diabetes mellitus. However, in the slowly progressive type, the degree of agreement of these two kits was poorer compared with those in all participants or in the acute type. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Measurement of activity for S-adenosylmethionine decarboxylase using radioisotope 14C

International Nuclear Information System (INIS)

Ko, Kyong Cheol; Park, Sang Hyun; Kamio, Yoshiyuku

2007-01-01

Polyamines are essential for normal cell growth and have important physiological function. They are polycationic compounds that are present in all biological materials. Also, they have been implicated in a wide variety of biological reactions. Generally, putrescine and spermidine are contained high amount in prokaryote, but spermidine and spermine are in eukaryote, respectively. However, S. ruminantium cells contain the polyamins such as spermidine and spermine. Addition of an aminopropyl group to putrescine conducts to the synthesis of spermidine. Aminopropyl group is derived from the dcSAM, a decarboxylation of S-adenosylmethionine, through action of S-adenosylmethionine decarboxylase (SAMDC). We suggested that S. ruminantium has a different pathway compare with prokaryote for polyamine synthesis. Assay for SAMDC activity was used 14 C labeled substrate. Key enzyme in the biosynthesis of polyamines, SAMDC, was purified from S. ruminantium and characterized. The enzyme was purified about 1,259-fold to electrophoretic homogeneity with a specific activity of 1.89×10 -5 kat kg'- 1 of protein

Structural Characterization of the Molecular Events during a Slow Substrate-Product Transition in Orotidine 5'-Monophosphate Decarboxylase

Energy Technology Data Exchange (ETDEWEB)

Fujihashi, Masahiro; Wei, Lianhu; Kotra, Lakshmi P; Pai, Emil F; (TGRI); (Toronto); (Kyoto)

2009-04-06

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.

Redox Cycling, pH Dependence, and Ligand Effects of Mn(III) in Oxalate Decarboxylase from Bacillus subtilis.

Science.gov (United States)

Twahir, Umar T; Ozarowski, Andrew; Angerhofer, Alexander

2016-11-29

This contribution describes electron paramagnetic resonance (EPR) experiments on Mn(III) in oxalate decarboxylase of Bacillus subtilis, an interesting enzyme that catalyzes the redox-neutral dissociation of oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized, although the N-terminal Mn(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions, in agreement with the general view that it represents the active site. Significantly, Mn(III) was observed in untreated OxDC in succinate and acetate buffers, while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR-visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn(III) are affected by small carboxylate ligands that can enter the active site and have been recorded for formate, acetate, and succinate. The results from a previous report [Zhu, W., et al. (2016) Biochemistry 55, 429-434] could therefore be reinterpreted as evidence of formate-bound Mn(III) after the enzyme is allowed to turn over oxalate. The pH dependence of the Mn(III) EPR signal compares very well with that of enzymatic activity, providing strong evidence that the catalytic reaction of oxalate decarboxylase is driven by Mn(III), which is generated in the presence of dioxygen.

EXPERIENCE OF ORNITHINE ASPARTATE (HEPA-MERZ AND PROBIOTICS BIOFLORUM FORTE IN THE TREATMENT OF NON-SEVERE FORMS OF ALCOHOLIC AND NON-ALCOHOLIC FATTY LIVER DISEASE

Directory of Open Access Journals (Sweden)

L. Yu. Ilchenko

2016-01-01

Full Text Available Aim: to evaluate the efficacy and tolerability of ornithine aspartate, probiotic Bioflorum Forte and their combination with steatosis and steatohepatitis in patients  with alcohol and non-alcoholic  fatty  liver disease. Materials and methods.  An open, randomized,  comparative  clinical study, which included 30 outpatients and inpatients with a diagnosis of steatosis, steatohepatitis. We analyzed the clinical symptoms, functional state of the liver. With the help of questionnaires  (Grids LeGo and post intoxication alcohol syndrome have established the presence of chronic alcohol intoxication. Test transmissions of numbers used to characterize the cognitive function, as well as detection  of minimal hepatic encephalopathy. Quality of life was assessed by questionnaire for patients with chronic liver disease — CLDQ (The chronic liver disease questionnaire. The duration of treatment was4 weeks. Results: all three treatment regimens have demonstrated therapeutic  efficacy: clinical improvement, recovery of liver function and results in cognitive function. When combined therapy also produced a significant improvement  in patients’ quality of life. It is shown that  the safety and tolerability of the means employed, adverse events were not reported. Conclusion: the results obtained allow us to recommend the use of ornithine aspartate (Hepa-Merz, both as monotherapy and as part of complex therapy of steatosis,  steatohepatitis with probiotic Bioflorum Forte in patients with alcoholic and non-alcoholic fatty liver disease.

Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone).

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Igarashi, K; Morris, D R

1984-11-01

Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.

Polyamine biosynthesis is critical for growth and differentiation of the pancreas

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Mastracci, Teresa L.; Robertson, Morgan A.; Mirmira, Raghavendra G.; Anderson, Ryan M.

2015-01-01

The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation. PMID:26299433

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

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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.

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

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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.

Acute treatment of hyperammonemia by continuous renal replacement therapy in a newborn patient with ornithine transcarbamylase deficiency

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Hyo Jeong Kim

2011-10-01

Full Text Available Ornithine transcarbamylase (OTC deficiency is well known as the most common inherited disorder of the urea cycle, and 1 of the most common causes of hyperammonemia in newborns. We experienced a case of a 3-day-old boy with OTC deficiency who appeared healthy in the first 2 days of life but developed lethargy and seizure soon afterwards. His serum ammonia level was measured as &gt;1700 μg/dL (range, 0 to 45 μg/dL. Continuous renal replacement therapy (CRRT in the mode of continuous venovenous hemodiafiltration was immediately applied to correct the raised ammonia level. No seizure occurred after the elevated ammonia level was reduced. Therefore, CRRT should be included as 1 of the treatment modalities for newborns with inborn errors of metabolism, especially hyperammonemia. Here, we report 1 case of successful treatment of hyperammonemia by CRRT in a neonate with OTC deficiency.

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

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Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

2016-01-15

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic

L-Dopa decarboxylase expression profile in human cancer cells.

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Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Thiol Redox Sensitivity of Two Key Enzymes of Heme Biosynthesis and Pentose Phosphate Pathways: Uroporphyrinogen Decarboxylase and Transketolase

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Brian McDonagh

2013-01-01

Full Text Available Uroporphyrinogen decarboxylase (Hem12p and transketolase (Tkl1p are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP. The redox properties of both Hem12p and Tkl1p from Saccharomyces cerevisiae were investigated using proteomic techniques (SRM and label-free quantification and biochemical assays in cell extracts and in vitro with recombinant proteins. The in vivo analysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2 in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae.

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Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong

2017-12-01

γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

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Streit, Bennett R; Celis, Arianna I; Shisler, Krista; Rodgers, Kenton R; Lukat-Rodgers, Gudrun S; DuBois, Jennifer L

2017-01-10

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O 2 and H 2 O 2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O 2 to the ferric state. The subsequent second-order reaction between the ferric complex and H 2 O 2 is slow, pH-dependent, and further decelerated by D 2 O 2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H 2 O 2 . Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H 2 O 2 cleavage is therefore unclear. From a cellular perspective, the use of H 2 O 2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

Autoantibodies against voltage-gated potassium channel and glutamic acid decarboxylase in psychosis: A systematic review, meta-analysis, and case series.

OpenAIRE

Grain, Rosemary; Lally, John; Stubbs, Brendon; Malik, Steffi; LeMince, Anne; Nicholson, Timothy R; Murray, Robin M; Gaughran, Fiona

2017-01-01

Antibodies to the voltage-gated potassium channel (VGKC) complex and glutamic acid decarboxylase (GAD) have been reported in some cases of psychosis. We conducted the first systematic review and meta-analysis to investigate their prevalence in people with psychosis and report a case series of VGKC-complex antibodies in refractory psychosis. Only five studies presenting prevalence rates of VGKC seropositivity in psychosis were identified, in addition to our case series, with an overall prevale...

Evidences for the agmatine involvement in antidepressant like effect of bupropion in mouse forced swim test.

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Kotagale, Nandkishor R; Tripathi, Sunil J; Aglawe, Manish M; Chopde, Chandrabhan T; Umekar, Milind J; Taksande, Brijesh G

2013-06-01

Although bupropion has been widely used in the treatment of depression, the precise mechanism of its therapeutic actions is not fully understood. The present study investigated the role of agmatine in an antidepressant like effect of bupropion in mouse forced swim test. The antidepressant like effect of bupropion was potentiated by pretreatment with agmatine (10-20mg/kg, ip) and by the drugs known to increase endogenous agmatine levels in brain viz., l-arginine (40 μg/mouse, icv), an agmatine biosynthetic precursor, ornithine decarboxylase inhibitor, dl-α-difluoromethyl ornithine hydrochloride, DFMO (12.5 μg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 μg/mouse, icv) and agmatinase inhibitor, arcaine (50 μg/mouse, icv) as well as imidazoline I1 receptor agonists, moxonidine (0.25mg/kg, ip) and clonidine (0.015 mg/kg, ip) and imidazoline I2 receptor agonist, 2-(2-benzofuranyl)-2-imidazoline hydrochloride, 2-BFI (5mg/kg, ip). Conversely, prior administration of I1 receptor antagonist, efaroxan (1mg/kg, ip) and I2 receptor antagonist, idazoxan (0.25mg/kg, ip) blocked the antidepressant like effect of bupropion and its synergistic combination with agmatine. These results demonstrate involvement of agmatine in the antidepressant like effect of bupropion and suggest agmatine and imidazoline receptors as a potential therapeutic target for the treatment of depressive disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

4-Amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) exerts in vitro growth inhibitory effects that are not only related to S-adenosylmethionine decarboxylase (SAMdc) inhibition

NARCIS (Netherlands)

Dorhout, B; Odink, MFG; deHoog, E; Kingma, AW; vanderVeer, E; Muskiet, FAJ

1997-01-01

The competitive S-adenosylmethionine decarboxylase (SAMdc; EC 4.1.1.50) inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) inhibits growth more effectively than the irreversible SAMdc inhibitor 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (AbeAdo), while having similar

Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine.

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Williams, Brianna B; Van Benschoten, Andrew H; Cimermancic, Peter; Donia, Mohamed S; Zimmermann, Michael; Taketani, Mao; Ishihara, Atsushi; Kashyap, Purna C; Fraser, James S; Fischbach, Michael A

2014-10-08

Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are largely unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrate that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of methylglyoxal bis(guanylhydrazone) and two phenylated analogues on S-adenosylmethionine decarboxylase activity from Eimeria stiedai (Apicomplexa).

Science.gov (United States)

San-Martín Núñez, B; Alunda, J M; Balaña-Fouce, R; Ordóñez Escudero, D

1987-01-01

1. Activity of S-adenosylmethionine decarboxylase, one of the rate-limiting enzymes of polyamine biosynthesis, was determined in oocysts of Eimeria stiedai, a coccidian parasite of the rabbit. 2. Several properties of the enzyme were compared to the mammalian enzyme. It showed considerably less substrate affinity than the analog enzyme from the rabbit. 3. The E. stiedai enzyme showed a low sensitivity to methylglyoxal bis(guanylhydrazone), a frequently used inhibitor of the enzyme in mammals, and two phenylated derivatives. 4. Results with the inhibitors are discussed in view of their potential use in chemotherapy.

A Dopa Decarboxylase Modulating the Immune Response of Scallop Chlamys farreri

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Zhou, Zhi; Yang, Jialong; Wang, Lingling; Zhang, Huan; Gao, Yang; Shi, Xiaowei; Wang, Mengqiang; Kong, Pengfei; Qiu, Limei; Song, Linsheng

2011-01-01

Background Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. Methodology The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, PDDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (PDDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc. PMID:21533240

Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

OpenAIRE

Jänne, J; Morris, D R

1984-01-01

Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

Glutamate decarboxylase and. gamma. -aminobutyric acid transaminase activity in brain structures during action of high concentrated sulfide gas on a background of hypo- and hypercalcemia

Energy Technology Data Exchange (ETDEWEB)

Kadyrov, G.K.; Aliyev, A.M.

Activity of the following enzymes was studied on the background of hypo- and hypercalcemia and exposure to high concentration of sulfide gas: glutamate decarboxylase (GDC) and {gamma}-aminobutyric acid transaminase (GABA-T). These enzymes regulate metabolism of GABA. The results showed that a 3.5 hr exposure to sulfide gas at a concentration of 0.3 mg/1 led to significantly increased activity of GDC in cerebral hemispheres, cerebellum and in brain stem. Activity of GABA-T dropped correspondingly. On the background of hypercalcemia induced by im. injection of 10% calcium gluconate (0.6 m1/200 g body weight of experimental rats) the negative effect caused by the exposure to sulfide gas was diminished. Under conditions of hypocalcemia (im. injection of 10 mg/200 g body weight of sodium oxalate), exposure to sulfide gas led to a significantly decreased activity of GDC and GABA-T in the hemispheres and in the brain stem, but in the cerebellum the activity of GDC increased sharply while that of GABA-T decreased correspondingly. 8 refs.

Ornithine aminotransferase (OAT): recombination between an X-linked OAT sequence (7.5 kb) and the Norrie disease locus.

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Ngo, J T; Bateman, J B; Spence, M A; Cortessis, V; Sparkes, R S; Kivlin, J D; Mohandas, T; Inana, G

1990-01-01

A human ornithine aminotransferase (OAT) locus has been mapped to the Xp11.2, as has the Norrie disease locus. We used a cDNA probe to investigate a 3-generation UCLA family with Norrie disease; a 4.2-kb RFLP was detected and a maximum lod score of 0.602 at zero recombination fraction was calculated. We used the same probe to study a second multigeneration family with Norrie disease from Utah. A different RFLP of 7.5 kb in size was identified and a recombinational event between the OAT locus represented by this RFLP and the disease loci was observed. Linkage analysis of these two loci in this family revealed a maximum load score of 1.88 at a recombination fraction of 0.10. Although both families have affected members with the same disease, the lod scores are reported separately because the 4.2- and 7.5-kb RFLPs may represent two different loci for the X-linked OAT.

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

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Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

2014-10-16

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target?

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Thales Kronenberger

2014-01-01

Full Text Available Malaria is a deadly infectious disease which affects millions of people each year in tropical regions. There is no effective vaccine available and the treatment is based on drugs which are currently facing an emergence of drug resistance and in this sense the search for new drug targets is indispensable. It is well established that vitamin biosynthetic pathways, such as the vitamin B6 de novo synthesis present in Plasmodium, are excellent drug targets. The active form of vitamin B6, pyridoxal 5-phosphate, is, besides its antioxidative properties, a cofactor for a variety of essential enzymes present in the malaria parasite which includes the ornithine decarboxylase (ODC, synthesis of polyamines, the aspartate aminotransferase (AspAT, involved in the protein biosynthesis, and the serine hydroxymethyltransferase (SHMT, a key enzyme within the folate metabolism.

Nigella sativa (black cumin) ameliorates potassium bromate-induced early events of carcinogenesis: diminution of oxidative stress.

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Khan, Naghma; Sharma, Sonia; Sultana, Sarwat

2003-04-01

Potassium bromate (KBrO3) is a potent nephrotoxic agent. In this paper, we report the chemopreventive effect of Nigella sativa (black cumin) on KBrO3-mediated renal oxidative stress, toxicity and tumor promotion response in rats. KBrO3 (125 mg/kg body weight, intraperitoneally) enhances lipid peroxidation, gamma-glutamyl transpeptidase, hydrogen peroxide and xanthine oxidase with reduction in the activities of renal antioxidant enzymes and renal glutathione content. A marked increase in blood urea nitrogen and serum creatinine has also been observed. KBrO3 treatment also enhances ornithine decarboxylase (ODC) activity and [3H] thymidine incorporation into renal DNA. Prophylaxis of rats orally with Nigella sativa extract (50 mg/kg body weight and 100 mg/kg body weight) resulted in a significant decrease in renal microsomal lipid peroxidation (P stress, toxicity and tumour promotion response in rats.

The effects of constant and alternating temperatures on the reproductive potential, life span, and life expectancy of Anastrepha fraterculus (Wiedemann (Dipteria: Tephritidae

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V. V. CARDOSO

Full Text Available Ovarian development, oviposition, larval eclosion, ornithine decarboxylase (ODC activity, ovarian, testis and ejaculatory apodeme measurements (length, width, and area, and the number of spermatozoa of Anastrepha fraterculus (Wiedemann were analyzed at alternating (20º/6ºC and 20º/13°C and constant (6°C; 25°C temperatures. Life span and life expectancy were also analyzed for both genders. All the results suggest that temperature, especially alternating temperatures, increase not only male and female reproductive potential but also their life span and life expectancy. These changes can be a powerful strategy triggered by A. fraterculus as a means to survive the stressful temperature conditions found in winter in the apple production region in Brazil, enabling this species to increase its population density and cause apple damage when spring begins.

The effects of constant and alternating temperatures on the reproductive potential, life span, and life expectancy of Anastrepha fraterculus (Wiedemann (Dipteria: Tephritidae

Directory of Open Access Journals (Sweden)

CARDOSO V. V.

2002-01-01

Full Text Available Ovarian development, oviposition, larval eclosion, ornithine decarboxylase (ODC activity, ovarian, testis and ejaculatory apodeme measurements (length, width, and area, and the number of spermatozoa of Anastrepha fraterculus (Wiedemann were analyzed at alternating (20masculine/6masculineC and 20masculine/13degreesC and constant (6degreesC; 25degreesC temperatures. Life span and life expectancy were also analyzed for both genders. All the results suggest that temperature, especially alternating temperatures, increase not only male and female reproductive potential but also their life span and life expectancy. These changes can be a powerful strategy triggered by A. fraterculus as a means to survive the stressful temperature conditions found in winter in the apple production region in Brazil, enabling this species to increase its population density and cause apple damage when spring begins.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

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Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

2017-10-12

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells1

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Miller, Aaron L; Johnson, Betty H; Medh, Rheem D; Townsend, Courtney M; Thompson, E Brad

2002-01-01

Abstract Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone (Dex) and two polyamine inhibitors, difluoromethylornithine (DFMO) and methyl glyoxal bis guanylhydrazone (MGBG), on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity. PMID:11922393

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells

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Aaron L. Miller

2002-01-01

Full Text Available Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone. (20Dex and two polyamine inhibitors, difluoromethylornithine. (20DFMO and methyl glyoxal bis guanylhydrazone. (20MGBG, on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase. (20ODC, the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity.

Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and -nonresponsive forms of gyrate atrophy

International Nuclear Information System (INIS)

Ramesh, V.; McClatchey, A.I.; Ramesh, N.; Benoit, L.A.; Berson, E.L.; Shih, V.E.; Gusella, J.F.

1988-01-01

Gyrate atrophy (GA), a recessive eye disease involving progressive loss of vision due to chorioretinal degeneration, is associated with a deficiency of the mitochondrial enzyme ornithine aminotransferase with consequent hyperornithinemia. Genetic heterogeneity of GA has been suggested by the demonstration that administration of pyridoxine to increase the level of pyridoxal phosphate, a cofactor of OATase, reduces hyperornithinemia in a subset of patients. The authors have cloned and sequences cDNAs for OATase from two GA patients, one responsive and one nonresponsive to pyridoxine treatment. The respective cDNAs contained different single missense mutations, which were sufficient to eliminate OATase activity when each cDNA was tested in a eukaryotic expression system. However, like the enzyme in fibroblasts from the pyridoxine-responsive patient, OATase encoded by the corresponding cDNA from this individual showed a significant increase in activity when assayed in the presence of an increased pyridoxal phosphate concentration. These data firmly establish that both pyridoxine responsive and nonresponsive forms of GA result from mutations in the OATase structural gene. Moreover, they provide a molecular characterization of the primary lesion in a pyridoxine-responsive genetic disorder

Autoantibodies against voltage-gated potassium channel (VGKC) and glutamic acid decarboxylase (GAD) in psychosis: A systematic review, meta-analysis and case series.

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Lally*, John; Grain*, Rosemary; Stubbs, Brendon; Malik, Steffi; LeMince, Anne; Nicholson, Timothy RJ; Murray, Robin MacGregor; Gaughran, Fiona Patricia

2017-01-01

Antibodies to the voltage-gated potassium channel (VGKC) complex and glutamic acid decarboxylase (GAD) have been reported in some cases of psychosis. We conducted the first systematic review and meta-analysis to investigate their prevalence in people with psychosis and report a case series of VGKC-complex antibodies in refractory psychosis. Only five studies presenting prevalence rates of VGKC seropositivity in psychosis were identified, in addition to our case series, with an overall prevale...

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

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Mehrotra nee Tandon, P; Lind, D S; Bear, H D; Susskind, B M

1992-08-01

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.

Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia.

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Gass, Nathalie; Glagotskaia, Tatiana; Mellema, Stefan; Stuurman, Jeroen; Barone, Mario; Mandel, Therese; Roessner-Tunali, Ute; Kuhlemeier, Cris

2005-08-01

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen-pistil interaction.

Measurement of activity for S-adenosylmethionine decarboxylase using radioisotope {sup 14}C

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Ko, Kyong Cheol; Park, Sang Hyun [Radiation Research Center for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Kamio, Yoshiyuku [Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University (Japan)

2007-05-15

Polyamines are essential for normal cell growth and have important physiological function. They are polycationic compounds that are present in all biological materials. Also, they have been implicated in a wide variety of biological reactions. Generally, putrescine and spermidine are contained high amount in prokaryote, but spermidine and spermine are in eukaryote, respectively. However, S. ruminantium cells contain the polyamins such as spermidine and spermine. Addition of an aminopropyl group to putrescine conducts to the synthesis of spermidine. Aminopropyl group is derived from the dcSAM, a decarboxylation of S-adenosylmethionine, through action of S-adenosylmethionine decarboxylase (SAMDC). We suggested that S. ruminantium has a different pathway compare with prokaryote for polyamine synthesis. Assay for SAMDC activity was used {sup 14}C labeled substrate. Key enzyme in the biosynthesis of polyamines, SAMDC, was purified from S. ruminantium and characterized. The enzyme was purified about 1,259-fold to electrophoretic homogeneity with a specific activity of 1.89×10{sup -5} kat kg'-{sup 1} of protein.

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

OpenAIRE

Gailey, Donald A.; Bordne, Deborah L.; Vallés, Ana Maria; Hall, Jeffrey C.; White, Kalpana

1987-01-01

An unstable Ring-X chromosome, Ddc+- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)wvC and a Rod-X chromosome which contained a P-element mediated Ddc + insert. The resulting Ddc+-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc+-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics reveal...

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

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Rusu Anca

2010-12-01

Full Text Available Abstract Background The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine and amino acids (e.g. arginine and ornithine are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied. Results Following inoculation of susceptible wheat heads by F. graminearum, DON accumulation was detected at two days after inoculation. The accumulation of putrescine was detected as early as one day following inoculation while arginine and cadaverine were also produced at three and four days post-inoculation. Transcripts of ornithine decarboxylase (ODC and arginine decarboxylase (ADC, two key biosynthetic enzymes for putrescine biosynthesis, were also strongly induced in heads at two days after inoculation. These results indicated that elicitation of the polyamine biosynthetic pathway is an early response to FHB. Transcripts for genes encoding enzymes acting upstream in the polyamine biosynthetic pathway as well as those of ODC and ADC, and putrescine levels were also induced in the rachis, a flower organ supporting DON production and an important route for pathogen colonisation during FHB. A survey of 24 wheat genotypes with varying responses to FHB showed putrescine induction is a general response to inoculation and no correlation was observed between the accumulation of putrescine and infection or DON accumulation. Conclusions The activation of the polyamine biosynthetic

Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells.

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Suzuki, T; Sadakata, Y; Kashiwagi, K; Hoshino, K; Kakinuma, Y; Shirahata, A; Igarashi, K

1993-07-15

A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

Developmental PCB Exposure Increases Audiogenic Seizures and Decreases Glutamic Acid Decarboxylase in the Inferior Colliculus.

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Bandara, Suren B; Eubig, Paul A; Sadowski, Renee N; Schantz, Susan L

2016-02-01

Previously, we observed that developmental polychlorinated biphenyl (PCB) exposure resulted in an increase in audiogenic seizures (AGSs) in rats. However, the rats were exposed to loud noise in adulthood, and were not tested for AGS until after 1 year of age, either of which could have interacted with early PCB exposure to increase AGS susceptibility. This study assessed susceptibility to AGS in young adult rats following developmental PCB exposure alone (without loud noise exposure) and investigated whether there was a decrease in GABA inhibitory neurotransmission in the inferior colliculus (IC) that could potentially explain this effect. Female Long-Evans rats were dosed orally with 0 or 6 mg/kg/day of an environmentally relevant PCB mixture from 28 days prior to breeding until the pups were weaned at postnatal day 21. One male-female pair from each litter was retained for the AGS study whilst another was retained for Western blot analysis of glutamic acid decarboxylase (GAD) and GABAAα1 receptor in the IC, the site in the auditory midbrain where AGS are initiated. There was a significant increase in the number and severity of AGSs in the PCB groups, with females somewhat more affected than males. GAD65 was decreased but there was no change in GAD67 or GABAAα1 in the IC indicating decreased inhibitory regulation in the PCB group. These results confirm that developmental PCB exposure alone is sufficient to increase susceptibility to AGS, and provide the first evidence for a possible mechanism of action at the level of the IC. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment

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Singh Sandeep

2012-09-01

Full Text Available Abstract Background GAD65 (Glutamic acid decarboxylase 65 KDa isoform is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription. Results The present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse β-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment. Conclusion We propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.

Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026.

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Zhang, Xiangmei; Wang, Zhangqian; Jan, Saad; Yang, Qian; Wang, Mo

2017-06-05

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

Refractory status epilepticus and glutamic acid decarboxylase antibodies in adults: presentation, treatment and outcomes.

Science.gov (United States)

Khawaja, Ayaz M; Vines, Brannon L; Miller, David W; Szaflarski, Jerzy P; Amara, Amy W

2016-03-01

Glutamic acid decarboxylase antibodies (GAD-Abs) have been implicated in refractory epilepsy. The association with refractory status epilepticus in adults has been rarely described. We discuss our experience in managing three adult patients who presented with refractory status epilepticus associated with GAD-Abs. Case series with retrospective chart and literature review. Three patients without pre-existing epilepsy who presented to our institution with generalized seizures between 2013 and 2014 were identified. Seizures proved refractory to first and second-line therapies and persisted beyond 24 hours. Patient 1 was a 22-year-old female who had elevated serum GAD-Ab titres at 0.49 mmol/l (normal: status epilepticus. Causation cannot be established since GAD-Abs may be elevated secondary to concurrent autoimmune diseases or formed de novo in response to GAD antigen exposure by neuronal injury. Based on this report and available literature, there may be a role for immuno- and chemotherapy in the management of refractory status epilepticus associated with GAD-Abs.

MRI findings in glutamic acid decarboxylase associated autoimmune epilepsy

Energy Technology Data Exchange (ETDEWEB)

Fredriksen, Jason R.; Carr, Carrie M.; Koeller, Kelly K.; Verdoorn, Jared T.; Kotsenas, Amy L. [Mayo Clinic, Department of Radiology, Rochester, MN (United States); Gadoth, Avi; Pittock, Sean J. [Mayo Clinic, Department of Neurology, Rochester, MN (United States)

2018-03-15

Glutamic acid decarboxylase (GAD65) has been implicated in a number of autoimmune-associated neurologic syndromes, including autoimmune epilepsy. This study categorizes the spectrum of MRI findings in patients with a clinical diagnosis of autoimmune epilepsy and elevated serum GAD65 autoantibodies. An institutional database search identified patients with elevated serum GAD65 antibodies and a clinical diagnosis of autoimmune epilepsy who had undergone brain MRI. Imaging studies were reviewed by three board-certified neuroradiologists and one neuroradiology fellow. Studies were evaluated for cortical/subcortical and hippocampal signal abnormality, cerebellar and cerebral volume loss, mesial temporal sclerosis, and parenchymal/leptomeningeal enhancement. The electronic medical record was reviewed for relevant clinical information and laboratory markers. A study cohort of 19 patients was identified. The majority of patients were female (84%), with a mean age of onset of 27 years. Serum GAD65 titers ranged from 33 to 4415 nmol/L (normal < 0.02 nmol/L). The most common presentation was medically intractable, complex partial seizures with temporal lobe onset. Parenchymal atrophy was the most common imaging finding (47%), with a subset of patients demonstrating cortical/subcortical parenchymal T2 hyperintensity (37%) or abnormal hippocampal signal (26%). No patients demonstrated abnormal parenchymal/leptomeningeal enhancement. The most common MRI finding in GAD65-associated autoimmune epilepsy is disproportionate parenchymal atrophy for age, often associated with abnormal cortical/subcortical T2 hyperintensities. Hippocampal abnormalities are seen in a minority of patients. This constellation of findings in a patient with medically intractable epilepsy should raise the possibility of GAD65 autoimmunity. (orig.)

Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli.

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McGregor, Wade C; Gillner, Danuta M; Swierczek, Sabina I; Liu, Dali; Holz, Richard C

2013-01-01

The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

Probiotic properties of lactic acid bacteria isolated from water-buffalo mozzarella cheese.

Science.gov (United States)

Jeronymo-Ceneviva, Ana Beatriz; de Paula, Aline Teodoro; Silva, Luana Faria; Todorov, Svetoslav Dimitrov; Franco, Bernadette Dora G Mello; Penna, Ana Lúcia B

2014-12-01

This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of β-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the β-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.

Antibacterial activity of oregano and sage plant extracts against decarboxylase-positive enterococci isolated from rabbit meat

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Ľubica Chrastinová

2013-02-01

Full Text Available The effect of plant extracts (sage, oregano against decarboxylase-positive enterococci from rabbit back limb meat  was reported in this study. Oregano plant extract inhibited the growth of all 34 tested enterococci (the inhibitory zones: 12 to 45 mm. The growth of the majority of strains  (n=23 was inhibited by oregano plant extract (the high size inhibitory zones (higher than 25 mm. The growth of 11 strains  was inhibited by oregano extract reaching medium size inhibitory zones (10 to 25mm. The most sensitive strain to oregano extract was E. faecium M7bA (45 mm. Sage extract was less active against tested enterococci (n=16  reaching lower inhibitory zones (up to 10 mm. doi:10.5219/239 Normal 0 21 false false false SK X-NONE X-NONE

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

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Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

Science.gov (United States)

Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Heat stress induced changes in metabolic regulators of donkeys from arid tracts in India

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Kataria N.

2012-05-01

Full Text Available To find out heat stress induced changes in metabolic regulators of donkeys from arid tracts in India, blood samples were collected to harvest the serum during moderate and extreme hot ambiences. The metabolic enzymes determined were sorbitol dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase, gammaglutamayl transferase, 5’nucleotidase, glucose-6-phosphatase, arginase, and aldolase. The mean values of all the serum enzymes increased significantly (p≤0.05 during hot ambience as compared to respective values during moderate ambience. It was concluded that increased activity of all the enzymes in the serum was due to modulation of metabolic reactions to combat the effect of hot ambience on the animals. Activation of gluconeogenesis along with hexose monophosphate shunt and urea cycle probably helped the animals to combat the heat stress.

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

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Katoch, Meenu; Mazmouz, Rabia; Chau, Rocky; Pearson, Leanne A.; Pickford, Russell

2016-01-01

ABSTRACT Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens. PMID:27520810

Isolation and characterization of xanthan-degrading Enterobacter sp. nov. LB37 for reducing the viscosity of xanthan in petroleum industry.

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Chen, Xiaoyi; Wang, Mi; Yang, Fan; Tang, Wenzhu; Li, Xianzhen

2014-05-01

A Gram-negative, straight rod and facultative anaerobic bacterium was isolated from soil sample. It exhibits the phenotypic characteristics consistent with its classification in the genus Enterobacter. The isolate ferment glucose to acid and gas. Arginine dihydrolase, ornithin decarboxylase and gelatinase but not deoxyribonuclease was produced by this isolate. There was no hydrogen sulfide production. On the basis of the phenotypic data, together with phylogenetic analysis based on 16S rDNA gene sequences, this strain should represent a novel species of the genus Enterobacter and was designated as LB37. The strain LB37 could degrade xanthan molecules resulting in the rapid decrease of the viscosity of xanthan solution used in oil drilling process. Endoxanthanase activity was also detected in the culture supernatant. To our knowledge, it is the first report on the microbes being involved in the xanthan degradation for oil industry. The isolate LB37 would be useful for potential application in enhanced oil recovery and oil drilling field.

Inhibiting cycloxygenase and ornithine decarboxylase by diclofenac and alpha-difluoromethylornithine blocks cutaneous SCCs by targeting Akt-ERK axis.

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Arumugam, Aadithya; Weng, Zhiping; Talwelkar, Sarang S; Chaudhary, Sandeep C; Kopelovich, Levy; Elmets, Craig A; Afaq, Farrukh; Athar, Mohammad

2013-01-01

Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser(473) & thr(308)) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.

Inhibiting cycloxygenase and ornithine decarboxylase by diclofenac and alpha-difluoromethylornithine blocks cutaneous SCCs by targeting Akt-ERK axis.

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Aadithya Arumugam

Full Text Available Non-melanoma skin cancer (NMSC is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser(473 & thr(308 were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.

Benchmarking pKa prediction methods for Lys115 in acetoacetate decarboxylase.

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Liu, Yuli; Patel, Anand H G; Burger, Steven K; Ayers, Paul W

2017-05-01

Three different pK a prediction methods were used to calculate the pK a of Lys115 in acetoacetate decarboxylase (AADase): the empirical method PROPKA, the multiconformation continuum electrostatics (MCCE) method, and the molecular dynamics/thermodynamic integration (MD/TI) method with implicit solvent. As expected, accurate pK a prediction of Lys115 depends on the protonation patterns of other ionizable groups, especially the nearby Glu76. However, since the prediction methods do not explicitly sample the protonation patterns of nearby residues, this must be done manually. When Glu76 is deprotonated, all three methods give an incorrect pK a value for Lys115. If protonated, Glu76 is used in an MD/TI calculation, the pK a of Lys115 is predicted to be 5.3, which agrees well with the experimental value of 5.9. This result agrees with previous site-directed mutagenesis studies, where the mutation of Glu76 (negative charge when deprotonated) to Gln (neutral) causes no change in K m , suggesting that Glu76 has no effect on the pK a shift of Lys115. Thus, we postulate that the pK a of Glu76 is also shifted so that Glu76 is protonated (neutral) in AADase. Graphical abstract Simulated abundances of protonated species as pH is varied.

Induction of the Histamine-Forming Enzyme Histidine Decarboxylase in Skeletal Muscles by Prolonged Muscular Work: Histological Demonstration and Mediation by Cytokines.

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Ayada, Kentaro; Tsuchiya, Masahiro; Yoneda, Hiroyuki; Yamaguchi, Kouji; Kumamoto, Hiroyuki; Sasaki, Keiichi; Tadano, Takeshi; Watanabe, Makoto; Endo, Yasuo

2017-01-01

Recent studies suggest that histamine-a regulator of the microcirculation-may play important roles in exercise. We have shown that the histamine-forming enzyme histidine decarboxylase (HDC) is induced in skeletal muscles by prolonged muscular work (PMW). However, histological analysis of such HDC induction is lacking due to appropriate anti-HDC antibodies being unavailable. We also showed that the inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α can induce HDC, and that PMW increases both IL-1α and IL-1β in skeletal muscles. Here, we examined the effects (a) of PMW on the histological evidence of HDC induction and (b) of IL-1β and TNF-α on HDC activity in skeletal muscles. By immunostaining using a recently introduced commercial polyclonal anti-HDC antibody, we found that cells in the endomysium and around blood vessels, and also some muscle fibers themselves, became HDC-positive after PMW. After PMW, TNF-α, but not IL-1α or IL-1β, was detected in the blood serum. The minimum intravenous dose of IL-1β that would induce HDC activity was about 1/10 that of TNF-α, while in combination they synergistically augmented HDC activity. These results suggest that PMW induces HDC in skeletal muscles, including cells in the endomysium and around blood vessels, and also some muscle fibers themselves, and that IL-1β and TNF-α may cooperatively mediate this induction.

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

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

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.

Effect of short-term ornithine alpha-ketoglutarate pretreatment on intestinal ischemia-reperfusion in rats Efeitos do pré-tratamento em curto prazo com ornitina alfa-cetoglutarato na isquemia-reperfusão intestinal em ratos

OpenAIRE

Eduardo Silvio Gouveia Gonçalves; Camila Menezes Rabelo; Alberico Ximenes do Prado Neto; José Huygens Parente Garcia; Sérgio Botelho Guimarães; Paulo Roberto Leitão de Vasconcelos

2011-01-01

PURPOSE: To investigate the effects of preventive enteral administration of ornithine alpha-ketoglutarate (OKG) in an ischemia-reperfusion rat model. METHODS: Sixty rats were randomized into five groups (G1-G5, n = 12). Each group was divided into two subgroups (n = 6) and treated with calcium carbonate (CaCa) or OKG by gavage. Thirty minutes later, the animals were anesthetized with xylazine 15mg + ketamine 1mg ip and subjected to laparotomy. G1-G3 rats served as controls. Rats in groups G4 ...

Norrie disease: linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe.

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Ngo, J T; Bateman, J B; Cortessis, V; Sparkes, R S; Mohandas, T; Inana, G; Spence, M A

1989-05-01

Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.

Reduced Functional Connectivity of Default Mode and Set-Maintenance Networks in Ornithine Transcarbamylase Deficiency.

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Ileana Pacheco-Colón

Full Text Available Ornithine transcarbamylase deficiency (OTCD is an X-chromosome linked urea cycle disorder (UCD that causes hyperammonemic episodes leading to white matter injury and impairments in executive functioning, working memory, and motor planning. This study aims to investigate differences in functional connectivity of two resting-state networks--default mode and set-maintenance--between OTCD patients and healthy controls.Sixteen patients with partial OTCD and twenty-two control participants underwent a resting-state scan using 3T fMRI. Combining independent component analysis (ICA and region-of-interest (ROI analyses, we identified the nodes that comprised each network in each group, and assessed internodal connectivity.Group comparisons revealed reduced functional connectivity in the default mode network (DMN of OTCD patients, particularly between the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC node and bilateral inferior parietal lobule (IPL, as well as between the ACC/mPFC node and the posterior cingulate cortex (PCC node. Patients also showed reduced connectivity in the set-maintenance network, especially between right anterior insula/frontal operculum (aI/fO node and bilateral superior frontal gyrus (SFG, as well as between the right aI/fO and ACC and between the ACC and right SFG.Internodal functional connectivity in the DMN and set-maintenance network is reduced in patients with partial OTCD compared to controls, most likely due to hyperammonemia-related white matter damage. Because several of the affected areas are involved in executive functioning, it is postulated that this reduced connectivity is an underlying cause of the deficits OTCD patients display in this cognitive domain.

Effect of short-term ornithine alpha-ketoglutarate pretreatment on intestinal ischemia-reperfusion in rats.

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Gonçalves, Eduardo Silvio Gouveia; Rabelo, Camila Menezes; Prado Neto, Alberico Ximenes do; Garcia, José Huygens Parente; Guimarães, Sérgio Botelho; Vasconcelos, Paulo Roberto Leitão de

2011-01-01

To investigate the effects of preventive enteral administration of ornithine alpha-ketoglutarate (OKG) in an ischemia-reperfusion rat model. Sixty rats were randomized into five groups (G1-G5, n = 12). Each group was divided into two subgroups (n = 6) and treated with calcium carbonate (CaCa) or OKG by gavage. Thirty minutes later, the animals were anesthetized with xylazine 15mg + ketamine 1mg ip and subjected to laparotomy. G1-G3 rats served as controls. Rats in groups G4 and G5 were subjected to ischemia for 30 minutes. Ischemia was achieved by clamping the small intestine and its mesentery, delimiting a segment of bowel 5 cm long and 5 cm apart from the ileocecal valve. In addition, G5 rats underwent reperfusion for 30 minutes. Blood samples were collected at the end of the laparotomy (G1), after 30 minutes (G2, G4) and 60 minutes (G3, G5) to determine concentrations of metabolites (pyruvate, lactate), creatine phosphokinase (CPK), thiobarbituric acid reactive substances (TBARS) and glutathione (GSH). There was a significant decrease in tissue pyruvate and lactate and plasma CPK levels in OKG-treated rats at the end of reperfusion period. GSH levels did not change significantly in ischemia and reperfusion groups. However, TBARS levels increased significantly (p<0.05) in tissue samples in OKG-treated rats subjected to ischemia for 30 minutes. Short-term pretreatment with OKG before induction of I/R decreases tissue damage, increases pyruvate utilization for energy production in the Krebs cycle and does not attenuate the oxidative stress in this animal model.

Glutamate decarboxylase immunoreactivity and gamma-[3H] aminobutyric acid accumulation within the same neurons in dissociated cell cultures of cerebral cortex

International Nuclear Information System (INIS)

Neale, E.A.; Oertel, W.H.; Bowers, L.M.; Weise, V.K.

1983-01-01

In order to evaluate the reliability of high affinity [ 3 H]GABA accumulation as a marker for GABAergic neurons, murine cerebral cortical neurons were studied in dissociated cell culture. Cultures which had been incubated in [ 3 H]GABA were stained immunohistochemically for the GABA-synthesizing enzyme, glutamate decarboxylase, fixed with paraformaldehyde, and subsequently processed for radioautography. In mature cultures, there was an 84 to 94% correlation between the presence of the enzyme and [ 3 H]GABA uptake within the same cortical neurons. These data provide direct evidence that those neurons which synthesize GABA are the same neurons which are labeled by high affinity [ 3 H]GABA uptake

Simultaneous silencing of two arginine decarboxylase genes alters development in Arabidopsis

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Diana eSánchez-Rangel

2016-03-01

Full Text Available Polyamines (PAs are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2 catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC. The generated transgenic lines (amiR:ADC-L1 and -L2 showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes.

Biotic and abiotic stress tolerance in transgenic tomatoes by constitutive expression of S-adenosylmethionine decarboxylase gene.

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Hazarika, Pranjal; Rajam, Manchikatla Venkat

2011-04-01

Recent findings have implicated the role of polyamines (putrescine, spermidine and spermine) in stress tolerance. Therefore, the present work was carried out with the goal of generating transgenic tomato plants with human S-adenosylmethionine decarboxylase (samdc) gene, a key gene involved in biosynthesis of polyamines, viz. spermidine and spermine and evaluating the transgenic plants for tolerance to both biotic and abiotic stresses. Several putative transgenic tomato plants with normal phenotype were obtained, and the transgene integration and expression was validated by PCR, Southern blot analysis and RT-PCR analysis, respectively. The transgenic plants exhibited high levels of polyamines as compared to the untransformed control plants. They also showed increased resistance against two important fungal pathogens of tomato, the wilt causing Fusarium oxysporum and the early blight causing Alternaria solani and tolerance to multiple abiotic stresses such as salinity, drought, cold and high temperature. These results suggest that engineering polyamine accumulation can confer tolerance to both biotic and abiotic stresses in plants.

Syndromic intellectual disability: a new phenotype caused by an aromatic amino acid decarboxylase gene (DDC) variant.

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Graziano, Claudio; Wischmeijer, Anita; Pippucci, Tommaso; Fusco, Carlo; Diquigiovanni, Chiara; Nõukas, Margit; Sauk, Martin; Kurg, Ants; Rivieri, Francesca; Blau, Nenad; Hoffmann, Georg F; Chaubey, Alka; Schwartz, Charles E; Romeo, Giovanni; Bonora, Elena; Garavelli, Livia; Seri, Marco

2015-04-01

The causative variant in a consanguineous family in which the three patients (two siblings and a cousin) presented with intellectual disability, Marfanoid habitus, craniofacial dysmorphisms, chronic diarrhea and progressive kyphoscoliosis, has been identified through whole exome sequencing (WES) analysis. WES study identified a homozygous DDC variant in the patients, c.1123C>T, resulting in p.Arg375Cys missense substitution. Mutations in DDC cause a recessive metabolic disorder (aromatic amino acid decarboxylase, AADC, deficiency, OMIM #608643) characterized by hypotonia, oculogyric crises, excessive sweating, temperature instability, dystonia, severe neurologic dysfunction in infancy, and specific abnormalities of neurotransmitters and their metabolites in the cerebrospinal fluid (CSF). In our family, analysis of neurotransmitters and their metabolites in patient's CSF shows a pattern compatible with AADC deficiency, although the clinical signs are different from the classic form. Our work expands the phenotypic spectrum associated with DDC variants, which therefore can cause an additional novel syndrome without typical movement abnormalities. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning and characterization of the ddc homolog encoding L-2,4-diaminobutyrate decarboxylase in Enterobacter aerogenes.

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Yamamoto, S; Mutoh, N; Tsuzuki, D; Ikai, H; Nakao, H; Shinoda, S; Narimatsu, S; Miyoshi, S I

2000-05-01

L-2,4-diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1,3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13048 was cloned and characterized. Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis. Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteriaceae. Such a relatively wide occurrence of the ddc homologs implies biological significance of DABA DC and its product DAP.

Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle.

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Yamashita, Tomoko; Nishimura, Kazuhiro; Saiki, Ryotaro; Okudaira, Hiroyuki; Tome, Mayuko; Higashi, Kyohei; Nakamura, Mizuho; Terui, Yusuke; Fujiwara, Kunio; Kashiwagi, Keiko; Igarashi, Kazuei

2013-06-01

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

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Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional Characterization of Waterlogging and Heat Stresses Tolerance Gene Pyruvate decarboxylase 2 from Actinidia deliciosa

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Hui-Ting Luo

2017-11-01

Full Text Available A previous report showed that both Pyruvate decarboxylase (PDC genes were significantly upregulated in kiwifruit after waterlogging treatment using Illumina sequencing technology, and that the kiwifruit AdPDC1 gene was required during waterlogging, but might not be required during other environmental stresses. Here, the function of another PDC gene, named AdPDC2, was analyzed. The expression of the AdPDC2 gene was determined using qRT-PCR, and the results showed that the expression levels of AdPDC2 in the reproductive organs were much higher than those in the nutritive organs. Waterlogging, NaCl, and heat could induce the expression of AdPDC2. Overexpression of kiwifruit AdPDC2 in transgenic Arabidopsis enhanced resistance to waterlogging and heat stresses in five-week-old seedlings, but could not enhance resistance to NaCl and mannitol stresses at the seed germination stage and in early seedlings. These results suggested that the kiwifruit AdPDC2 gene may play an important role in waterlogging resistance and heat stresses in kiwifruit.

Expression and Bioinformatic Analysis of Ornithine Aminotransferase 
in Non-small Cell Lung Cancer

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Danfei ZHOU

2012-09-01

Full Text Available Background and objective It has been proven that ornithine aminotransferase (OAT might play an important role in the oncogenesis and progression of numerous malignant tumors. The aim of this study is to detect the mRNA and protein expression of OAT in non-small cell lung cancer (NSCLC, as well as to analyze the bioinformatic features and binary interactions. Methods OAT mRNA expression was detected in A549 and 16HBE cell lines by reverse transcription-polymerase chain reaction. OAT protein expression was determined in 55 cases of NSCLC and 17 cases of adjacent non-tumor lung tissues by immunohistochemical staining. The bioinformatic features and binary interactions of OAT were analyzed. Gene ontology annotation and signal pathway analysis were performed. Results OAT mRNA expression in A549 cells was 2.85-fold lower than that in 16HBE cells. OAT protein expression was significantly higher in NSCLC tissues than that in adjacent non-tumor lung tissues. A significant difference of OAT protein expression was existed between squamous cell lung cancer and adenocarcinoma (P<0.05, but was not correlated with the gender, age, lymph node metastasis, tumor size, and TNM stages. Bioinformatic analysis suggested that OAT was a highly homologous and stable protein located in the mitochondria. An aminotran-3 domain and several sites of phosphorylation, which may function in signal transduction, gene transcription, and molecular transit, were found. In the 54 selected binary interactions of OAT, TNF and TRAF6 play roles in the NF-κB pathway. Conclusion OAT may play an important role in the oncogenesis and progression of NSCLC. Thus, OAT may be a novel biomarker for the diagnosis of NSCLC or a new target for its treatment.

S-adenosylmethionine decarboxylase inhibitors: new aryl and heteroaryl analogues of methylglyoxal bis(guanylhydrazone).

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Stanek, J; Caravatti, G; Capraro, H G; Furet, P; Mett, H; Schneider, P; Regenass, U

1993-01-08

A series of 3-acylbenzamidine (amidino)hydrazones 7a-h, the corresponding (hetero)aromatic congeners 7i-p, and 3,3'-bis-amidino-biaryls 25a-e were synthesized. The hydrazones 7a-p were prepared by conversion of the corresponding acyl nitriles 1a,c-d,i,n-p to the imido esters 3a,c-d,i and the amidines 5a,c-d,h-i, followed by a reaction with aminoguanidine, or vice versa. Similarly, the biaryl 3,3'-dinitriles 23a-e were converted, via the imino esters 24a-c or the imino thioesters 27d-e, to the diamidines 25a-e. These new products are conformationally constrained analogues of methylglyoxal bis(guanylhydrazone) (MGBG). They are up to 100 times more potent as inhibitors of rat liver S-adenosylmethionine decarboxylase (SMDC) and generally less potent inhibitors of rat small intestine diamine oxidase (DAO) than MGBG. Some of these SAMDC inhibitors, e.g., compounds 7a, 7e, 7i, 25a, and 25d, have shown antiproliferative effects against T24 human bladder carcinoma cells. These products, whose structure-activity relationships are discussed, are of interest as potential anticancer agents and drugs for the treatment of protozoal and Pneumocystis carinii infections.

Expression analysis and clinical utility of L-Dopa decarboxylase (DDC) in prostate cancer.

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Avgeris, Margaritis; Koutalellis, Georgios; Fragoulis, Emmanuel G; Scorilas, Andreas

2008-10-01

L-Dopa decarboxylase (DDC) is a pyridoxal 5'-phosphate-dependent enzyme that was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of DDC in prostate tissues and to evaluate its clinical utility in prostate cancer (CaP). Total RNA was isolated from 118 tissue specimens from benign prostate hyperplasia (BPH) and CaP patients and a highly sensitive quantitative real-time RT-PCR (qRT-PCR) method for DDC mRNA quantification has been developed using the SYBR Green chemistry. LNCaP prostate cancer cell line was used as a calibrator and GAPDH as a housekeeping gene. DDC was found to be overexpressed, at the mRNA level, in the specimens from prostate cancer patients, in comparison to those from benign prostate hyperplasia patients (pDDC expression has significant discriminatory value between CaP and BPH (pDDC expression status was compared with other established prognostic factors, in prostate cancer. High expression levels of DDC were found more frequently in high Gleason's score tumors (p=0.022) as well as in advanced stage patients (p=0.032). Our data reveal the potential of DDC expression, at the mRNA level, as a novel biomarker in prostate cancer.

Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

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Shuhei Noda

Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells

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Giancarlo eRusso

2014-12-01

Full Text Available The function of the enzyme glutamate decarboxylase (GAD is to convert glutamate in -aminobutyric acid (GABA.GAD exists as two major isoforms, termed GAD65 and GAD67,.that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

Agmatine Production by Aspergillus oryzae is Elevated by Low pH During Solid-State Cultivation.

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Akasaka, Naoki; Kato, Saori; Kato, Saya; Hidese, Ryota; Wagu, Yutaka; Sakoda, Hisao; Fujiwara, Shinsuke

2018-05-25

Sake (rice wine) produced by multiple parallel fermentation (MPF) involving Aspergillus oryzae (strain RW) and Saccharomyces cerevisiae under solid-state cultivation conditions contained 3.5 mM agmatine, while that produced from enzymatically saccharified rice syrup by S. cerevisiae contained oryzae under solid-state cultivation (3.1 mM) but not under submerged cultivation, demonstrating that A. oryzae in solid-state culture produces agmatine. The effect of cultivation conditions on agmatine production was examined. Agmatine production was boosted at 30°C and reached the highest level (6.3 mM) at pH 5.3. The addition of l-lactic, succinic, and citric acids reduced the initial culture pH to 3.0, 3.5, and 3.2, respectively, resulting in further increase in agmatine accumulation (8.2, 8.7, and 8.3 mM, respectively). Homogenate from a solid-state culture exhibited a maximum l-arginine decarboxylase (ADC) activity (74 pmol min -1 μg -1 ) at pH 3.0 at 30°C; that from a submerged culture exhibited an extremely low activity (oryzae , even though A. oryzae lacks ADC orthologs and, instead, possesses four ornithine decarboxylases (ODC1-4). Recombinant ODC1 and ODC2 exhibited no ADC activity at acidic pH (pH 4.0>), suggesting that other decarboxylases or an unidentified ADC is involved in agmatine production. IMPORTANCE It has been speculated that, in general, fungi do not synthesize agmatine from l-arginine because they do not possess genes encoding for arginine decarboxylase. Numerous preclinical studies have shown that agmatine exerts pleiotropic effects on various molecular targets, leading to an improved quality of life. In the present study, we first demonstrated that l-arginine was a feasible substrate for agmatine production by the fungus Aspergillus oryzae RW. We observed that the productivity of agmatine by A. oryzae RW was elevated at low pH only during solid-state cultivation. A. oryzae is utilized in the production of various oriental fermented foods. The

Increases in urea synthesis and the ornithine-urea cycle capacity in the giant African snail, Achatina fulica, during fasting or aestivation, or after the injection with ammonium chloride.

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Hiong, Kum Chew; Loong, Ai May; Chew, Shit Fun; Ip, Yuen Kwong

2005-12-01

The objectives of this study are to determine whether a full complement of ornithine-urea cycle (OUC) enzymes is present in the hepatopancreas of the giant African snail Achatina fulica, and to investigate whether the rate of urea synthesis and the OUC capacity can be up-regulated during 23 days of fasting or aestivation, or 24 hr post-injection with NH(4)Cl (10 micromol g(-1) snail) into the foot muscle. A. fulica is ureotelic and a full complement of OUC enzymes, including carbamoyl phosphate synthetase III (CPS III), was detected from its hepatopancreas. There were significant increases in the excretion of NH(4)(+), NH(3) and urea in fasting A. fulica. Fasting had no significant effect on the tissue ammonia contents, but led to a progressive accumulation of urea, which was associated with an 18-fold increase in the rate of urea synthesis. Because fasting took place in the presence of water and because there was no change in water contents in the foot muscle and hepatopancreas, it can be concluded that the function of urea accumulation in fasting A. fulica was unrelated to water retention. Aestivation in arid conditions led to a non-progressive accumulation of urea in A. fulica. During the first 4 days and the last 3 days of the 23-day aestivation period, experimental snails exhibited significantly greater rates of urea synthesis compared with fasted snails. These increases were associated with significant increases in activities of various OUC enzymes, except CPS III, in the hepatopancreas. However, the overall urea accumulation in snails aestivated and snails fasted for 23 days were comparable. Therefore, the classical hypothesis that urea accumulation occurred to prevent water loss through evaporation during aestivation in terrestrial pulmonates may not be valid. Surprisingly, there were no accumulations of ammonia in the foot muscle and hepatopancreas of A. fulica 12 or 24 hr after NH(4)Cl was injected into the foot muscle. In contrast, the urea content in

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

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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.

Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina

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Ishida, Yuko; Ozaki, Mamiko

2012-01-01

In the blowfly Phormia regina, exposure to d-limonene for 5 days during feeding inhibits proboscis extension reflex behavior due to decreasing tyramine (TA) titer in the brain. TA is synthesized by tyrosine decarboxylase (Tdc) and catalyzed into octopamine (OA) by TA ß-hydroxylase (Tbh). To address the mechanisms of TA titer regulation in the blowfly, we cloned Tdc and Tbh cDNAs from P. regina (PregTdc and PregTbh). The deduced amino acid sequences of both proteins showed high identity to those of the corresponding proteins from Drosophila melanogaster at the amino acid level. PregTdc was expressed in the antenna, labellum, and tarsus whereas PregTbh was expressed in the head, indicating that TA is mainly synthesized in the sensory organs whereas OA is primarily synthesized in the brain. d-Limonene exposure significantly decreased PregTdc expression in the antenna but not in the labellum and the tarsus, indicating that PregTdc expressed in the antenna is responsible for decreasing TA titer. PregTdc-like immunoreactive material was localized in the thin-walled sensillum. In contrast, the OA/TA receptor (PregOAR/TAR) was localized to the thick-walled sensillum. The results indicated that d-limonene inhibits PregTdc expression in the olfactory receptor neurons in the thin-walled sensilla, likely resulting in reduced TA levels in the receptor neurons in the antenna. TA may be transferred from the receptor neuron to the specific synaptic junction in the antennal lobe of the brain through the projection neurons and play a role in conveying the aversive odorant information to the projection and local neurons.

Fatal ammonia toxicity in an adult due to an undiagnosed urea cycle defect: under-recognition of ornithine transcarbamylase deficiency.

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Thurlow, Vanessa R; Asafu-Adjaye, Michelle; Agalou, Stamatina; Rahman, Yusof

2010-05-01

There is a lack of awareness of acutely presenting inborn errors of metabolism in adults, of which the X-linked urea cycle defect ornithine transcarbamylase (OTC) deficiency is an example, many comparatively mild mutations having been identified. In male hemizygotes clinical manifestations and age at presentation vary and depend on the mutation. In female heterozygotes the clinical spectrum depends on the extent to which the abnormal gene is expressed. Milder versions of the defect may not cause clear clinical symptoms and may remain unrecognized until the person is subjected to an unusually high nitrogen load when they develop severe hyperammonaemia. During acute episodes liver enzymes may be normal or only slightly elevated and occasionally accompanied by coagulopathy, but the key finding is hyperammonaemia. Boys with these milder forms may exhibit abnormal behaviour and be diagnosed with attention deficit hyperactivity disorder. This case illustrates how late presentation of OTC deficiency in a non-specialist centre can be difficult to differentiate from drug abuse, psychiatric illness or encephalopathy. Failure to measure blood ammonia in adults with unexplained key symptoms - particularly prolonged vomiting without diarrhoea and altered mental state/hallucinations, or to recognize the significance of elevated blood ammonia without evidence of liver decompensation can lead to delayed or missed diagnosis.

Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

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Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

2003-05-01

Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

L-dopa decarboxylase (DDC) gene expression is related to outcome in patients with prostate cancer.

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Koutalellis, Georgios; Stravodimos, Konstantinos; Avgeris, Margaritis; Mavridis, Konstantinos; Scorilas, Andreas; Lazaris, Andreas; Constantinides, Constantinos

2012-09-01

What's known on the subject? and What does the study add? L-dopa decarboxylase (DDC) has been documented as a novel co-activator of androgen receptor transcriptional activity. Recently, it was shown that DDC gene expression is significantly higher in patients with PCa than in those with BPH. In the present study, there was a significant association between the DDC gene expression levels and the pathological stage and Gleason score of patients with prostate cancer (PCa). Moreover, DDC expression was shown to be an unfavourable prognostic marker of biochemical recurrence and disease-free survival in patients with PCa treated by radical prostatectomy. To determine whether L-dopa decarboxylase gene (DDC) expression levels in patients with prostate cancer (PCa) correlate to biochemical recurrence and disease prognosis after radical prostatectomy (RP). The present study consisted of 56 samples with confirmed malignancy from patients with PCa who had undergone RP at a single tertiary academic centre. Total RNA was isolated from tissue specimens and a SYBR Green fluorescence-based quantitative real-time polymerase chain reaction methodology was developed for the determination of DDC mRNA expression levels of the tested tissues. Follow-up time ranged between 1.0 and 62.0 months (mean ± SE, 28.6 ± 2.1 month; median, 31.5 months). Time to biochemical recurrence was defined as the interval between the surgery and the measurement of two consecutive values of prostate-specific antigen (PSA) ≥0.2 ng/mL. DDC expression levels were found to be positively correlated with the tumour-node-metastasis stage (P = 0.021) and Gleason score (P = 0.036) of the patients with PCa. Patients with PCa with raised DDC expression levels run a significantly higher risk of biochemical recurrence after RP, as indicated by Cox proportional regression analysis (P = 0.021). Multivariate Cox proportional regression models revealed the preoperative PSA-, age- and digital rectal examination

Efficacy of l-ornithine-l-aspartate as an adjuvant therapy in cirrhotic patients with hepatic encephalopathy

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Abid, S.; Jafri, W.; Mumtaz, K.; Islam, M.; Abbas, Z.

2011-01-01

To evaluate the efficacy of L-ornithine-L-aspartate (LOLA) as an adjuvant therapy in cirrhotic patients with hepatic encephalopathy (HE). Study Design: Randomized placebo controlled study. Place and Duration of Study: The Aga Khan University Hospital, Karachi in the year 2003-2004. Methodology: Patients with HE were randomized to receive LOLA or placebo medicine as an adjuvant to treatment of HE. Number connection test-A (NCT-A), ammonia level, clinical grade of HE and duration of hospitalization were assessed. Results: Out of 120 patients, there were 62 males with mean age of 57 +- 11 years. Improvement in HE was higher (n=40, 66.7%) in LOLA group as compared to the placebo group (n=28, 46.7%, p=0.027). In patients with grade I or less encephalopathy, improvement was seen in 6 (35.3%) and 3 (20%) patients in LOLA and placebo groups respectively (p=0.667). Patients with HE grade II and above showed improvement in 34 (79.1%) and 25 (55.6%) cases in LOLA and placebo group respectively (p=0.019). On multivariate analysis patients with HE of grade II and above showed prothrombin time, creatinine level and use of LOLA influencing the outcome. Duration of hospitalization was 93.6 +- 25.7 hours and 135.2 +- 103.5 hours in LOLA and placebo groups respectively (p=0.025). No side effects were observed in either groups. Conclusion: In cirrhotic patients with advanced hepatic encephalopathy treatment with LOLA was safe and associated with relatively rapid improvement and shorter hospital stay. (author)

The clinical significance of detecting serum glutamic acid decarboxylase antibody (GAD), C-peptide and insulin in diabetics

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Zheng Tingliang; Zhang Jinchi; Yao Yingfei; Chen Linxing; Huang Hua

2005-01-01

Objective: To explore the clinical significance of detecting serum glutamic acid decarboxylase (GAD) antibody, C-peptide (CP) and insulin (INS) in the classification of diabetic patients. Methods: Serum GAD antibody, CP and INS concentration were determined with RIA in 27 patients with type 1 diabetes mellitus (DM1) and 49 patients with type 2 diabetes mellitus (DM2). Sugar-electrode-method was used to detect the concentrations of fasting plasma glucose (FPG) in these patients. Results: The positive rate of GAD antibody in DM1 patients (66.7%) were significantly higher than that in DM2 group (8.2%) (P<0.01), The levels of CP and INS were lower in DM1 group than those in DM2 group as well (P<0.01). Conclusion: GAD antibody is a valuable marker to predict the impairment of β-cell GAD antibody levels, together with CP /FPG and INS/FPG ratios, might be useful in determining the type of DM and guiding the therapy. (authors)

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

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Masalova, O V; Lesnova, E I; Samokhvalov, E I; Permyakova, K Yu; Ivanov, A V; Kochetkov, S N; Kushch, A A

2017-01-01

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

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Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de Las Rivas, Blanca; Muñoz, Rosario

2017-04-01

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase ( lpdC , or lp_2945 ) is only 6.5 kb distant from the gene encoding inducible tannase ( L. plantarum tanB [ tanB Lp ], or lp_2956 ). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B ( lpdB , or lp_0271 ) and D ( lpdD , or lp_0272 ) of the gallate decarboxylase are cotranscribed, whereas subunit C ( lpdC , or lp_2945 ) is cotranscribed with a gene encoding a transport protein ( gacP , or lp_2943 ). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator ( lp_2942 ) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are

Structure-function relations in oxaloacetate decarboxylase complex. Fluorescence and infrared approaches to monitor oxomalonate and Na(+ binding effect.

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Thierry Granjon

Full Text Available BACKGROUND: Oxaloacetate decarboxylase (OAD is a member of the Na(+ transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na(+ ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of alpha, beta and gamma subunits. The alpha subunit contains the carboxyltransferase catalytic site. METHODOLOGY/PRINCIPAL FINDINGS: In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate and Na(+ effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES, indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na(+. REES changes due to oxomalonate binding were also observed with the alphagamma and alpha subunits. Infrared spectra showed that OAD, alphagamma and alpha subunits have a main component band centered between 1655 and 1650 cm(-1 characteristic of a high content of alpha helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of beta sheet structures and a concomitant increase of alpha helix structures. Oxomalonate binding to alphagamma and alpha subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. CONCLUSION: Oxomalonate binding affects the

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions.

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Liang, Jing; Han, Qian; Ding, Haizhen; Li, Jianyong

2017-12-01

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO 2 , NH 3, and H 2 O 2 . This contrasts to the typical DDC-catalyzed reaction, which produces CO 2 and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H 2 O 2 in the process. Biochemical assessment established that H 2 O 2 , formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H 2

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa

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McCurtain, Jennifer L.; Gilbertsen, Adam J.; Kalstabakken, Kyle A.; Williams, Bryan J.

2018-01-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled 13C5,15N4-agmatine (synthesized by decarboxylation of uniformly labeled 13C6,15N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,l,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1 % (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients. PMID:25957842

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

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Dalluge, Joseph J; McCurtain, Jennifer L; Gilbertsen, Adam J; Kalstabakken, Kyle A; Williams, Bryan J

2015-07-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

Plackett-Burman and Box-Behnken designs as chemometric tools for micro-determination of L-Ornithine

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Elazazy, Marwa S.; El-Hamshary, Marwa; Sakr, Marwa; Al-Easa, Hala S.

2018-03-01

Plackett-Burman (PB) and Box-Behnken (BB) screening and response surface factorial designs were used to evaluate spectrophotometric and spectrofluorimetric approaches for the determination of L-Ornithine (ORN) as per se and in dietary supplements. Both approaches were based on the derivatization of the primary amino group of ORN via Hantzsch condensation reaction producing yellow coloured adducts (dihydrolutidine derivative). The reaction product was determined spectrophotometrically (method A) at λmax = 327 nm and spectrofluorimetrically (method B) at 480 nm (λem) after excitation at 325 nm (λex). A multivariate scheme was tailored to investigate the process numerical variables; reaction temperature, heating time, reagent volume, and pH implementing PB as a screening design followed by BB as an optimization strategy. Categorical factors including diluting solvent and sequence of addition were kept invariable. Responses of the reaction systems were the maximum absorbance (Y1) and maximum fluorescence intensity (Y2), correspondingly. Quality tools as well as ANOVA testing, before and after response transformation were used to decide upon the substantial variables. Following the optimization of reaction variables using desirability plots, calibration graphs were found to be rectilinear in the range of 6-14 μg/mL and 0.4-1.2 μg/mL for methods A and B, respectively. Both methods proved to be sensitive with detection limits (DL) of 337 and 85 ng/mL, and quantitation limits (QL) of 1086 and 283 ng/mL, for methods A and B, respectively. An interference study was performed using potential foreign species. No significant interference effect was observed on any of the proposed procedures. System performance was addressed following ICH guidelines and considering parameters such as linearity, detection and quantification limits, accuracy and precision, robustness and specificity.

Polyamines mediate abnormal Ca2+ transport and Ca2+-induced cardiac cell injury in the calcium paradox

International Nuclear Information System (INIS)

Trout, J.J.; Koenig, H.; Goldstone, A.D.; Lu, C.Y.; Fan, C.C.

1986-01-01

Ca 2+ -free perfusion renders heart cells Ca 2+ -sensitive so that readmission of Ca 2+ causes a sudden massive cellular injury attributed to abnormal entry of Ca 2+ into cells (Ca paradox). Hormonal stimulation of Ca 2+ fluxes was earlier shown to be mediated by polyamines (PA). 5 min perfusion of rat heart with Ca 2+ -free medium induce a prompt 40-50% decline in levels of the PA putrescine (PUT), spermidine and spermine and their rate-regulatory synthetic enzyme ornithine decarboxylase (ODC), and readmission of Ca 2+ -containing medium abruptly ( 2+ reperfusion-induced increases in ODC and PA and also prevented increased 45 Ca 2+ uptake and heart injury, manifested by loss of contractility, release of enzymes (CPK, LDH), myoglobin and protein, and E.M. lesions (contracture bands, mitochondrial changes). 1 mM PUT negated DFMO inhibition, repleted heart PA and restored Ca 2+ reperfusion-induced 45 Ca 2+ influx and cell injury. These data indicate that the Ca 2+ -directed depletion-repletion cycle of ODC and PA triggers excessive transsarcolemmal Ca 2+ transport leading to the calcium paradox

Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

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Igarashi, K; Porter, C W; Morris, D R

1984-11-01

Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

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Morris, D R; Jorstad, C M; Seyfried, C E

1977-09-01

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

Nucleotide variation at the dopa decarboxylase (Ddc) gene in natural populations of Drosophila melanogaster.

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Tatarenkov, Andrey; Ayala, Francisco J

2007-08-01

We studied nucleotide sequence variation at the gene coding for dopa decarboxylase (Ddc) in seven populations of Drosophila melanogaster. Strength and pattern of linkage disequilibrium are somewhat distinct in the extensively sampled Spanish and Raleigh populations. In the Spanish population, a few sites are in strong positive association, whereas a large number of sites in the Raleigh population are associated nonrandomly but the association is not strong. Linkage disequilibrium analysis shows presence of two groups of haplotypes in the populations, each of which is fairly diverged, suggesting epistasis or inversion polymorphism. There is evidence of two forms of natural selection acting on Ddc. The McDonald-Kreitman test indicates a deficit of fixed amino acid differences between D. melanogaster and D. simulans, which may be due to negative selection. An excess of derived alleles at high frequency, significant according to the H-test, is consistent with the effect of hitchhiking. The hitchhiking may have been caused by directional selection downstream of the locus studied, as suggested by a gradual decrease of the polymorphism-to-divergence ratio. Altogether, the Ddc locus exhibits a complicated pattern of variation apparently due to several evolutionary forces. Such a complex pattern may be a result of an unusually high density of functionally important genes.

Exogenous Application of GABA Improves PEG-Induced Drought Tolerance Positively Associated with GABA-Shunt, Polyamines, and Proline Metabolism in White Clover.

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Yong, Bin; Xie, Huan; Li, Zhou; Li, Ya-Ping; Zhang, Yan; Nie, Gang; Zhang, Xin-Quan; Ma, Xiao; Huang, Lin-Kai; Yan, Yan-Hong; Peng, Yan

2017-01-01

In order to investigate the physiological effects of exogenous γ-aminobutyric acid (GABA) on drought tolerance in white clover (Trifolium repens), GABA shunt, polyamines (PAs), and proline (Pro) metabolism were examined after plants pretreated with or without GABA (8 mM) and then exposed to water or 15% PEG-induced drought stress in growth chamber. In this study, exogenous application of GABA effectively alleviated drought-induced damage in leaves, as reflected by significantly higher relative water content, lower electrolyte leakage, lipid peroxidation, and leaf wilt. Exogenous GABA further promoted drought-induced increases in GABA transaminase and alpha ketone glutarate dehydrogenase activities, but inhibited glutamate decarboxylase activity under both control and drought conditions, resulting in an increase in endogenous glutamate (Glu) and GABA content. Besides, exogenous GABA could well accelerated PAs synthesis and suppressed PAs catabolism, which lead to the extremely enhanced different types of PAs content (free Put and Spd, insoluble bound Spd and Spm, soluble conjugated Spd and Spm, and total Put, Spd and Spm) under drought stress. In addition, exogenous GABA application further activated drought-induced Δ 1 -pyrroline-5-carboxylate synthetase and proline dehydrogenase activities, but suppressed drought-facilitated ornithine -δ-amino transferase activities, leading to a higher Pro accumulation and metabolism in GABA-pretreated plants in the middle and last period of drought. The results suggested that increased endogenous GABA by exogenous GABA treatment could improve drought tolerance of white clover associated with a positive regulation in the GABA-shunt, PAs and Pro metabolism.

Suppression of LFA-1 expression by spermine is associated with enhanced methylation of ITGAL, the LFA-1 promoter area.

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Yoshihiko Kano

Full Text Available Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1 expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt activity, enhanced demethylation of the LFA-1 gene (ITGAL promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM, and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM-such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase and SAM supplementation-successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL.

The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

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Tamara Kakoschke

Full Text Available To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin.

Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

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Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

2015-01-15

Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae

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Hideki Katow

2013-12-01

The ontogenetic origin of blastocoelar glutamate decarboxylase (GAD-expressing cells (GADCs in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells.

New and highly sensitive assay for L-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography-voltammetry.

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Rahman, M K; Nagatsu, T; Kato, T

1980-12-12

This paper describes a new, inexpensive and highly sensitive assay for aromatic L-amino acid decarboxylase (AADC) activity, using L-5-hydroxytryptophan (L-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. L-5-HTP was used as substrate and D-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from L-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using L-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma

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Jing Lin

2017-12-01

Full Text Available Self-renewing tumor-initiating cells (TICs are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC. GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5–7 nM, disrupt the open reading frame (ORF of GLDC transcript (predisposing it for nonsense-mediated decay, halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32. One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

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Fredrick, Chase M; Lin, Guangyun; Johnson, Eric A

2017-07-01

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD 600 ]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium. IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

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Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

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Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

2015-05-19

Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii.

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Lu, Zichun J; Markham, George D

2004-01-02

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.

Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

International Nuclear Information System (INIS)

Rosenberg, R.M.; O'Leary, M.H.

1985-01-01

The authors have measured the 13 C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D 2 O at pH 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D 2 O at pH 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13 C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. The authors have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13 C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier

Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

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Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo

2014-12-17

The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

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Di Bartolomeo, Francesca; Doan, Kim Nguyen; Athenstaedt, Karin; Becker, Thomas; Daum, Günther

2017-07-01

In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a β-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. Here, we present a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzyme's activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction of Polyamines, Abscisic Acid, Nitric Oxide, and Hydrogen Peroxide under Chilling Stress in Tomato (Lycopersicon esculentum Mill.) Seedlings.

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Diao, Qiannan; Song, Yongjun; Shi, Dongmei; Qi, Hongyan

2017-01-01

Polyamines (PAs) play a vital role in the responses of higher plants to abiotic stresses. However, only a limited number of studies have examined the interplay between PAs and signal molecules. The aim of this study was to elucidate the cross-talk among PAs, abscisic acid (ABA), nitric oxide (NO), and hydrogen peroxide (H 2 O 2 ) under chilling stress conditions using tomato seedlings [( Lycopersicon esculentum Mill.) cv. Moneymaker]. The study showed that during chilling stress (4°C; 0, 12, and 24 h), the application of spermidine (Spd) and spermine (Spm) elevated NO and H 2 O 2 levels, enhanced nitrite reductase (NR), nitric oxide synthase (NOS)-like, and polyamine oxidase activities, and upregulated LeNR relative expression, but did not influence LeNOS1 expression. In contrast, putrescine (Put) treatment had no obvious impact. During the recovery period (25/15°C, 10 h), the above-mentioned parameters induced by the application of PAs were restored to their control levels. Seedlings pretreated with sodium nitroprusside (SNP, an NO donor) showed elevated Put and Spd levels throughout the treatment period, consistent with increased expression in leaves of genes encoding arginine decarboxylase ( LeADC. LeADC1 ), ornithine decarboxylase ( LeODC ), and Spd synthase ( LeSPDS ) expressions in tomato leaves throughout the treatment period. Under chilling stress, the Put content increased first, followed by a rise in the Spd content. Exogenously applied SNP did not increase the expression of genes encoding S -adenosylmethionine decarboxylase ( LeSAMDC ) and Spm synthase ( LeSPMS ), consistent with the observation that Spm levels remained constant under chilling stress and during the recovery period. In contrast, exogenous Put significantly increased the ABA content and the 9- cis -epoxycarotenoid dioxygenase ( LeNCED1 ) transcript level. Treatment with ABA could alleviate the electrolyte leakage (EL) induced by D-Arg (an inhibitor of Put). Taken together, it is

Putrescine treatment reverses α-tocopherol-induced desynchronization of polyamine and retinoid metabolism during rat liver regeneration

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Lourdes Sánchez-Sevilla

2016-10-01

Full Text Available Abstract Background The pre-treatment with α-tocopherol inhibits progression of rat liver proliferation induced by partial hepatectomy (PH, by decreasing and/or desynchronizing cyclin D1 expression and activation into the nucleus, activation and nuclear translocation of STAT-1 and -3 proteins and altering retinoid metabolism. Interactions between retinoic acid and polyamines have been reported in the PH-induced rat liver regeneration. Therefore, we evaluated the effect of low dosage of α-tocopherol on PH-induced changes in polyamine metabolism. Methods This study evaluated the participation of polyamine synthesis and metabolism during α-tocopherol-induced inhibition of rat liver regeneration. In PH-rats (Wistar treated with α-tocopherol and putrescine, parameters indicative of cell proliferation, lipid peroxidation, ornithine decarboxylase expression (ODC, and polyamine levels, were determined. Results Pre-treatment with α-tocopherol to PH-animals exerted an antioxidant effect, shifting earlier the increased ODC activity and expression, temporally affecting polyamine synthesis and ornithine metabolism. Whereas administration of putrescine induced minor changes in PH-rats, the concomitant treatment actually counteracted most of adverse actions exerted by α-tocopherol on the remnant liver, restituting its proliferative potential, without changing its antioxidant effect. Putrescine administration to these rats was also associated with lower ODC expression and activity in the proliferating liver, but the temporally shifting in the amount of liver polyamines induced by α-tocopherol, was also “synchronized” by the putrescine administration. The latter is supported by the fact that a close relationship was observed between fluctuations of polyamines and retinoids. Conclusions Putrescine counteracted most adverse actions exerted by α-tocopherol on rat liver regeneration, restoring liver proliferative potential and restituting the decreased

Aromatic Amino Acid Decarboxylase Deficiency Not Responding to Pyridoxine and Bromocriptine Therapy: Case Report and Review of Response to Treatment

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Majid Alfadhel

2014-01-01

Full Text Available 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.

PPARα autocrine regulation of Ca²⁺-regulated exocytosis in guinea pig antral mucous cells: NO and cGMP accumulation.

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Tanaka, Saori; Sugiyama, Nanae; Takahashi, Yuko; Mantoku, Daiki; Sawabe, Yukinori; Kuwabara, Hiroko; Nakano, Takashi; Shimamoto, Chikao; Matsumura, Hitoshi; Marunaka, Yoshinori; Nakahari, Takashi

2014-12-15

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N(5)-[imino(propylamino)methyl]-L-ornithine hydrochloride (N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca(2+)-regulated exocytosis in antral mucous cells. Copyright © 2014 the American Physiological Society.

Loss of autonoetic consciousness of recent autobiographical episodes and accelerated long-term forgetting in a patient with previously unrecognized glutamic acid decarboxylase antibody related limbic encephalitis

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Juri-Alexander eWitt

2015-06-01

Full Text Available 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 consciousness regarding autobiographical memories of the last three 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 six 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

Cloning and Expression Vector Construction of Glutamate Decarboxylase Gene from Lactobacillus Plantarum

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B Arabpour

2016-06-01

Full Text Available BACKGROUND AND OBJECTIVE: Gamma-aminobutyric acid (GABA is a four-carbon non-protein amino acid used in the treatment of hypertension, diabetes, inflammation, and depression. GABA is synthesized by glutamic acid decarboxylase (GAD enzyme in many organisms, including bacteria. Therefore, cloning of this enzyme is essential to the optimization of GABA production. This study aimed to clone and construct the expression vector of GAD gene from Lactobacillus plantarum PTCC 1058 bacterium. METHODS: In this experimental study, we investigated the morphological, biochemical, genetic and 16s rDNA sequencing of L. plantarum PTCC 1058 strain. Genomic DNA of the bacterium was isolated and amplified using the GAD gene via polymerase chain reaction (PCR. Afterwards, the gene was inserted into the pJET1.2/blunt cloning vector and subcloned in vector pET32a. Plasmid pET32a-gad expression vector was transformed in Escherichia coli BL21 strain, and protein expression was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. FINDINGS: Morphological, biochemical and genetic analyses of 16s rDNA sequencing indicated that the studied substrain was of the L. plantarum strain. In addition, results of nucleotide sequencing of the fragmented segment via PCR showed the presence of GAD gene. Results of colony PCR and SDS-PAGE analysis confirmed the accuracy of the cloning and gene expression of the recombinant Escherichia coli BL21 strain. CONCLUSION: According to the results of this study, cloning of GAD gene from L. plantarum PTCC 1058 was successful. These cloned genes could grow rapidly in prokaryotic and eukaryotic systems and be used in cost-effective culture media and even non-recyclable waste.

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

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Yan Huang

Full Text Available 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.

Glutamic acid decarboxylase autoantibody-positivity post-partum is associated with impaired β-cell function in women with gestational diabetes mellitus

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Lundberg, T. P.; Højlund, K.; Snogdal, L. S.

2015-01-01

AIMS: To investigate whether the presence of glutamic acid decarboxylase (GAD) autoantibodies post-partum in women with prior gestational diabetes mellitus was associated with changes in metabolic characteristics, including β-cell function and insulin sensitivity. METHODS: During 1997-2010, 407...... women with gestational diabetes mellitus were offered a 3-month post-partum follow-up including anthropometrics, serum lipid profile, HbA1c and GAD autoantibodies, as well as a 2-h oral glucose tolerance test (OGTT) with blood glucose, serum insulin and C-peptide at 0, 30 and 120 min. Indices of insulin...... similar age and prevalence of diabetes mellitus. Women who were GAD+ve had significantly higher 2-h OGTT glucose concentrations during their index-pregnancy (10.5 vs. 9.8 mmol/l, P = 0.001), higher fasting glucose (5.2 vs. 5.0 mmol/l, P = 0.02) and higher 2-h glucose (7.8 vs. 7.1 mmol/l, P = 0.05) post...

Lack of Association Between Polymorphisms in Dopa Decarboxylase and Dopamine Receptor-1 Genes With Childhood Autism in Chinese Han Population.

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Yu, Hong; Liu, Jun; Yang, Aiping; Yang, Guohui; Yang, Wenjun; Lei, Heyue; Quan, Jianjun; Zhang, Zengyu

2016-04-01

Genetic factors play an important role in childhood autism. This study is to determine the association of single-nucleotide polymorphisms in dopa decarboxylase (DDC) and dopamine receptor-1 (DRD1) genes with childhood autism, in a Chinese Han population. A total of 211 autistic children and 250 age- and gender-matched healthy controls were recruited. The severity of disease was determined by Children Autism Rating Scale scores. TaqMan Probe by real-time polymerase chain reaction was used to determine genotypes and allele frequencies of single-nucleotide polymorphism rs6592961 in DDC and rs251937 in DRD1. Case-control and case-only studies were respectively performed, to determine the contribution of both single-nucleotide polymorphisms to the predisposition of disease and its severity. Our results showed that there was no significant association of the genotypes and allele frequencies of both single-nucleotide polymorphisms concerning childhood autism and its severity. More studies with larger samples are needed to corroborate their predicting roles. © The Author(s) 2015.

Screening and kinetics of glutaminase and glutamate decarboxylase producing lactic acid bacteria from fermented Thai foods

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Sasimar Woraharn

2014-12-01

Full Text Available L-glutaminase and glutamic acid decarboxylase (GAD catalyzes the hydrolysis of L-glutamine and glutamate, respectively. L-glutaminase widely used in cancer therapy along with a combination of other enzymes and most importantly these enzymes were used in food industries, as a major catalyst of bioconversion. The current investigation was aimed to screen and select L-glutaminase, and GAD producing lactic acid bacteria (LAB. A total of 338 LAB were isolated from fermented meat, fermented fish, fermented soya bean, fermented vegetables and fruits. Among 338 isolates, 22 and 237 LAB has been found to be positive for L-glutaminase and GAD, respectively. We found that 30 days of incubation at 35 ºC and pH 6.0 was the optimum condition for glutaminase activity by G507/1. G254/2 was found to be the best for GAD activity with the optimum condition of pH 6.5, temperature 40 ºC and ten days of incubation. These LAB strains, G507/1 and G254/2, were identified as close relative of Lactobacillus brevis ATCC 14869 and Lactobacillus fermentum NBRC 3956, respectively by 16S rRNA sequencing. Further, improvements in up-stream of the fermentation process with these LAB strains are currently under development.

Histidine Decarboxylase Knockout Mice as a Model of the Pathophysiology of Tourette Syndrome and Related Conditions.

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Pittenger, Christopher

2017-01-01

While the normal functions of histamine (HA) in the central nervous system have gradually come into focus over the past 30 years, the relationship of abnormalities in neurotransmitter HA to human disease has been slower to emerge. New insight came with the 2010 description of a rare nonsense mutation in the biosynthetic enzyme histidine decarboxylase (Hdc) that was associated with Tourette syndrome (TS) and related conditions in a single family pedigree. Subsequent genetic work has provided further support for abnormalities of HA signaling in sporadic TS. As a result of this genetic work, Hdc knockout mice, which were generated more than 15 years ago, have been reexamined as a model of the pathophysiology of TS and related conditions. Parallel work in these KO mice and in human carriers of the Hdc mutation has revealed abnormalities in the basal ganglia system and its modulation by dopamine (DA) and has confirmed the etiologic, face, and predictive validity of the model. The Hdc-KO model thus serves as a unique platform to probe the pathophysiology of TS and related conditions, and to generate specific hypotheses for subsequent testing in humans. This chapter summarizes the development and validation of this model and recent and ongoing work using it to further investigate pathophysiological changes that may contribute to these disorders.

Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase.

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Okai, Naoko; Takahashi, Chihiro; Hatada, Kazuki; Ogino, Chiaki; Kondo, Akihiko

2014-01-01

Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L(-1) glucose and 0.1 mM pyridoxal 5'-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L(-1) (0.259 g L(-1) h(-1)) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol(-1).

Common Variation in the DOPA Decarboxylase (DDC) Gene and Human Striatal DDC Activity In Vivo.

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Eisenberg, Daniel P; Kohn, Philip D; Hegarty, Catherine E; Ianni, Angela M; Kolachana, Bhaskar; Gregory, Michael D; Masdeu, Joseph C; Berman, Karen F

2016-08-01

The synthesis of multiple amine neurotransmitters, such as dopamine, norepinephrine, serotonin, and trace amines, relies in part on DOPA decarboxylase (DDC, AADC), an enzyme that is required for normative neural operations. Because rare, loss-of-function mutations in the DDC gene result in severe enzymatic deficiency and devastating autonomic, motor, and cognitive impairment, DDC common genetic polymorphisms have been proposed as a source of more moderate, but clinically important, alterations in DDC function that may contribute to risk, course, or treatment response in complex, heritable neuropsychiatric illnesses. However, a direct link between common genetic variation in DDC and DDC activity in the living human brain has never been established. We therefore tested for this association by conducting extensive genotyping across the DDC gene in a large cohort of 120 healthy individuals, for whom DDC activity was then quantified with [(18)F]-FDOPA positron emission tomography (PET). The specific uptake constant, Ki, a measure of DDC activity, was estimated for striatal regions of interest and found to be predicted by one of five tested haplotypes, particularly in the ventral striatum. These data provide evidence for cis-acting, functional common polymorphisms in the DDC gene and support future work to determine whether such variation might meaningfully contribute to DDC-mediated neural processes relevant to neuropsychiatric illness and treatment.

Cecembia lonarensis gen. nov., sp. nov., a haloalkalitolerant bacterium of the family Cyclobacteriaceae, isolated from a haloalkaline lake and emended descriptions of the genera Indibacter, Nitritalea and Belliella.

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Anil Kumar, P; Srinivas, T N R; Madhu, S; Sravan, R; Singh, Shashi; Naqvi, S W A; Mayilraj, S; Shivaji, S

2012-09-01

A novel Gram-staining-negative, rod-shaped, non-motile bacterium, designated strain LW9(T), was isolated from a water sample collected from Lonar Lake of Buldhana district, Maharashtra, India. Colonies and broth cultures were reddish orange due to the presence of carotenoid pigments. Strain LW9(T) was positive for catalase, ornithine decarboxylase and lysine decarboxylase activities and negative for gelatinase, oxidase, urease and lipase activities. The predominant fatty acids were iso-C(15 : 0) (31.3 %), iso-C(16 : 0) (9.3 %), anteiso-C(15 : 0) (7.3 %), iso-C(16 : 1) H (6.1 %), summed feature 3 (comprising C(16 : 1)ω7c/C(16 : 1)ω6c; 5.9 %), iso-C(17 : 1)ω9c (5.4 %) and iso-C(17 : 0) 3-OH (5.0 %). Strain LW9(T) contained MK-7 as the major respiratory quinone. The polar lipids consisted of phosphatidylethanolamine, two unidentified aminolipids and seven unidentified lipids. The DNA G+C content of strain LW9(T) was 40.5 mol%. 16S rRNA gene sequence analysis indicated that the type strains of Indibacter alkaliphilus and Aquiflexum balticum, two members of the family Cyclobacteriaceae (phylum 'Bacteroidetes') were the most closely related strains with sequence similarities of 93.0 and 94.0 %, respectively. Other members of the family Cyclobacteriaceae showed sequence similarities <93.0 %. Based on these phenotypic characteristics and on phylogenetic inference, strain LW9(T) is proposed as the representative of novel species in a new genus, Cecembia lonarensis gen. nov., sp. nov. The type strain of the type species, Cecembia lonarensis, is LW9(T) (= CCUG 58316(T) = KCTC 22772(T)). Emended descriptions of the genera Indibacter, Nitritalea and Belliella are also proposed.

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata.

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Liu, Wanhong; Chen, Rong; Chen, Min; Zhang, Haoxing; Peng, Meifang; Yang, Chunxian; Ming, Xingjia; Lan, Xiaozhong; Liao, Zhihua

2012-07-01

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K (m) of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K (m) of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g(-1) DW) than the EtOH control (0.183 mg g(-1) DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g(-1) DW) followed by in bark (0.161 mg g(-1) DW) and young leaf (0.130 mg g(-1) DW), and least in root (0.014 mg g(-1) DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.

Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

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Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

2012-09-17

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

Glutamic acid decarboxylase antibodies are indicators of the course, but not of the onset, of diabetes in middle-aged adults: the Atherosclerosis Risk in Communities Study

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A. Vigo

2007-07-01

Full Text Available To efficiently examine the association of glutamic acid decarboxylase antibody (GADA positivity with the onset and progression of diabetes in middle-aged adults, we performed a case-cohort study representing the ~9-year experience of 10,275 Atherosclerosis Risk in Communities Study participants, initially aged 45-64 years. Antibodies to glutamic acid decarboxylase (GAD65 were measured by radioimmunoassay in 580 incident diabetes cases and 544 non-cases. The overall weighted prevalence of GADA positivity (³1 U/mL was 7.3%. Baseline risk factors, with the exception of smoking and interleukin-6 (P £ 0.02, were generally similar between GADA-positive and -negative individuals. GADA positivity did not predict incident diabetes in multiply adjusted (HR = 1.04; 95%CI = 0.55, 1.96 proportional hazard analyses. However, a small non-significant adjusted risk (HR = 1.29; 95%CI = 0.58, 2.88 was seen for those in the highest tertile (³2.38 U/mL of positivity. GADA-positive and GADA-negative non-diabetic individuals had similar risk profiles for diabetes, with central obesity and elevated inflammation markers, aside from glucose, being the main predictors. Among diabetes cases at study's end, progression to insulin treatment increased monotonically as a function of baseline GADA level. Overall, being GADA positive increased risk of progression to insulin use almost 10 times (HR = 9.9; 95%CI = 3.4, 28.5. In conclusion, in initially non-diabetic middle-aged adults, GADA positivity did not increase diabetes risk, and the overall baseline profile of risk factors was similar for positive and negative individuals. Among middle-aged adults, with the possible exception of those with the highest GADA levels, autoimmune pathophysiology reflected by GADA may become clinically relevant only after diabetes onset.

Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

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Caldarera, C M; Casti, A; Guarnier, C; Moruzzi, G

1975-10-01

The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription.

Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

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Brendan T. McKeown

2015-01-01

Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

Long-term exposure to microwave radiation provokes cancer growth: evidences from radars and mobile communication systems.

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Yakymenko, I; Sidorik, E; Kyrylenko, S; Chekhun, V

2011-06-01

In this review we discuss alarming epidemiological and experimental data on possible carcinogenic effects of long term exposure to low intensity microwave (MW) radiation. Recently, a number of reports revealed that under certain conditions the irradiation by low intensity MW can substantially induce cancer progression in humans and in animal models. The carcinogenic effect of MW irradiation is typically manifested after long term (up to 10 years and more) exposure. Nevertheless, even a year of operation of a powerful base transmitting station for mobile communication reportedly resulted in a dramatic increase of cancer incidence among population living nearby. In addition, model studies in rodents unveiled a significant increase in carcinogenesis after 17-24 months of MW exposure both in tumor-prone and intact animals. To that, such metabolic changes, as overproduction of reactive oxygen species, 8-hydroxi-2-deoxyguanosine formation, or ornithine decarboxylase activation under exposure to low intensity MW confirm a stress impact of this factor on living cells. We also address the issue of standards for assessment of biological effects of irradiation. It is now becoming increasingly evident that assessment of biological effects of non-ionizing radiation based on physical (thermal) approach used in recommendations of current regulatory bodies, including the International Commission on Non-Ionizing Radiation Protection (ICNIRP) Guidelines, requires urgent reevaluation. We conclude that recent data strongly point to the need for re-elaboration of the current safety limits for non-ionizing radiation using recently obtained knowledge. We also emphasize that the everyday exposure of both occupational and general public to MW radiation should be regulated based on a precautionary principles which imply maximum restriction of excessive exposure.

Detection of autoantibodies against reactive oxygen species modified glutamic acid decarboxylase-65 in type 1 diabetes associated complications

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Mashal Subhash N

2011-03-01

Full Text Available Abstract Background Autoantibodies against glutamate decarboxylase-65 (GAD65Abs are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD65Abs are a sensitive and specific marker for type 1 diabetes (T1D. These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D, as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD65Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD65 in these three different GAD65Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD65 (ROS-GAD65 is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD65 (ROS-GAD65Abs and quantitative assays in T1D associated complications. Results From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD65 as compared to native GAD65 (N-GAD65. Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD65. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10-6 M followed by nephropathic (1.81 × 10-6 M and uncomplicated (3.11 × 10-7 M T1D patients for ROS-GAD65 compared to N-GAD65. Conclusion Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD65 that induce increased production of ROS-GAD65Abs. Hence regulation of ROS

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

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Oud Bart

2012-09-01

Full Text Available Abstract Background Pyruvate-decarboxylase negative (Pdc- strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v ethanol at a maximum specific growth rate (0.24 h-1 similar to that of the evolved Pdc- strain (0.23 h-1. Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1 than the evolved strain (0.20 h-1. The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae.

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Oud, Bart; Flores, Carmen-Lisset; Gancedo, Carlos; Zhang, Xiuying; Trueheart, Joshua; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

2012-09-15

Pyruvate-decarboxylase negative (Pdc⁻) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc⁻S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc⁻ strains cannot grow on high glucose concentrations and require C₂-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Genetic analysis of a Pdc⁻ strain previously evolved to overcome these deficiencies revealed a 225 p in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc⁻ strain enabled growth on 20 g l⁻¹ glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h⁻¹) similar to that of the evolved Pdc⁻ strain (0.23 h⁻¹). Furthermore, the reverse engineered Pdc⁻ strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h⁻¹) than the evolved strain (0.20 h⁻¹). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc⁻S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc⁻ strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C₂-compound auxotrophy. In this study we have discovered and characterised a

The effects of a "low-risk" diet on cell proliferation and enzymatic parameters of preneoplastic rat colon.

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Goettler, D; Rao, A V; Bird, R P

1987-01-01

The relationship between various dietary constituents and colon cancer has been demonstrated by previous research. This study was conducted to investigate the combined effects of several dietary constituents on the preneoplastic stage of azoxymethane (AOM)-induced colon cancer in rats. A nutritionally adequate, "low-risk" (LR) diet was formulated through the modulation of dietary fat, fiber, protein, vitamins A and E, and selenium. Female F344 rats were given three weekly subcutaneous injections of AOM and were maintained on either the LR diet or a "high-risk" (HR) diet. After 12 weeks, the rats were killed and the following parameters were determined: pH of colon contents, fecal beta-glucuronidase activity, tissue ornithine decarboxylase (ODC) activity, and colonic labeling index. The pH of the colon contents and incremental labeling index were lower in the group given the LR diet and treated with AOM compared with the group given the HR diet and treated with AOM; however, no statistically significant dietary effects were observed for beta-glucuronidase and ODC activities. The results of this study indicated that the colons of rats fed the LR diet exhibited different proliferative characteristics than did the colons of rats fed the HR diet.

Polyamines modulate carcinogen-induced mutagenesis in vivo.

Science.gov (United States)

Wallon, U Margaretha; O'Brien, Thomas G

2005-01-01

Elevated polyamine levels as a consequence of targeted overexpression of ornithine decarboxylase (ODC) to murine skin enhance susceptibility to tumorigenesis in this tissue. A possible mechanism for the enhanced susceptibility phenotype is an increased sensitivity of tissues with elevated polyamine levels to the mutagenic action of carcinogens. To test this hypothesis, a transgenic mouse model containing the Big Blue transgene and also expressing a K6/ODC transgene was developed. Incorporation of the K6/ODC transgene into the Big Blue model did not affect the spontaneous lacI mutant frequency in either skin or epidermis of the double-transgenic mice. After skin treatment with single doses of either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine, however, the mutant frequency was significantly increased in the skin of double-transgenic Big Blue;K6/ODC mice compared to Big Blue controls. The increases in mutant frequency were clearly due to ODC transgene activity, since treatment of mice with the ODC inhibitor, alpha-difluoromethylornithine, completely abolished the difference in mutant frequencies between double-transgenic and Big Blue mice. These results demonstrate that intracellular polyamine levels modulate mutation induction following carcinogen exposure. 2004 Wiley-Liss, Inc.

A Comprehensive Review of Punica granatum (Pomegranate) Properties in Toxicological, Pharmacological, Cellular and Molecular Biology Researches

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Rahimi, Hamid Reza; Arastoo, Mohammad; Ostad, Seyed Nasser

2012-01-01

Punica granatum (Pg), commonly known as pomegranate (Pg), is a member of the monogeneric family, Punicaceae, and is mainly found in Iran which is considered to be its primary centre of origin. Pg and its chemical components possess various pharmacological and toxicological properties including antioxidant, anti-inflammatory (by inhibiting pro-inflammatory cytokines), anti-cancer and anti-angiogenesis activities. They also show inhibitory effects on invasion/motility, cell cycle, apoptosis, and vital enzymes such as cyclooxygenase (COX), lipooxygenase (LOX), cytochrome P450 (CYP450), phospholipase A2 (PLA2), ornithine decarboxylase (ODC), carbonic anhydrase (CA), 17beta-hydroxysteroid dehydrogenase (17β-HSDs) and serine protease (SP). Furthermore, they can stimulate cell differentiation and possess anti-mutagenic effects. Pg can also interfere with several signaling pathways including PI3K/AKT, mTOR, PI3K, Bcl-X, Bax, Bad, MAPK, ERK1/2, P38, JNK, and caspase. However, the exact mechanisms for its pharmacological and toxicological properties remain to be unclear and need further evaluation. These properties strongly suggest a wide range use of Pg for clinical applications. This review will discuss the areas for which Pg has shown therapeutic properties in different mechanisms. PMID:24250463

Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.

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Taxis, Christof

2017-01-01

In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.

Sequence Classification: 778807 [

Lifescience Database Archive (English)

Full Text Available shifting from yeast to mammals (9.3 kD) (1G671) || http://www.ncbi.nlm.nih.gov/protein/17511075 ... ...hine decarboxylase antizyme, involved in polyamine regulation and subject to a conserved translational frame

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.

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Stribny, Jiri; Romagnoli, Gabriele; Pérez-Torrado, Roberto; Daran, Jean-Marc; Querol, Amparo

2016-03-12

The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The

Occurrence of Type 1 Diabetes in Graves' Disease Patients Who Are Positive for Antiglutamic Acid Decarboxylase Antibodies: An 8-Year Followup Study

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Matsuo Taniyama

2011-01-01

Full Text Available Glutamic acid decarboxylase antibodies (GADAs are one of the markers of islet cell autoimmunity and are sometimes present before the onset of type 1 diabetes (T1D. GADA can be present in Graves' patients without diabetes; however, the outcome of GADA-positive Graves' patients is not fully understood, and the predictive value of GADA for the development of T1D in Graves' patients remains to be clarified. We investigated the prevalence of GADA in 158 patients with Graves' disease and detected GADA in 10 patients. They were followed up to discover whether or not T1D developed. In the course of eight years, 2 patients with high titers of GADA developed T1D, both had long-standing antithyroid drug-resistant Graves' disease. Thus, Graves' disease with high GADA titer seems to be at high risk for T1D.

Organic synthesis with stable isotopes

International Nuclear Information System (INIS)

Blazer, R.M.; Daub, G.H.; Kerr, V.N.; Williams, D.L.; Whaley, T.W.

1982-01-01

Described is a scheme for the synthesis of L-arginine-1- 13 C utilizing methods developed for the synthesis of L-ornithine-1- 13 C from L-ornithine-2- 13 C and then converting ornithine into arginine with the enzyme acylase

Mass transfer ranking of polylysine, poly-ornithine and poly-methylene-co-guanidine microcapsule membranes using a single low molecular mass marker

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Rosinski Stefan

2003-01-01

Full Text Available On the long way to clinical transplantable hybrid systems, comprising of cells, acting as immuno-protected bioreactors microencapsulated in a polymeric matrix and delivering desired factors (proteins, hormones, enzymes etc to the patient's body, an important step is the optimization of the microcapsule. This topic includes the selection of a proper coating membrane which could fulfil, first of all, the mass transfer as well as biocompatibility, stability and durability requirements. Three different membranes from polymerised aminoacids, formed around exactly identical alginate gel cores, were considered, concerning their mass transport properties, as potential candidates in this task. The results of the evaluation of the mass ingress and mass transfer coefficient h for the selected low molecular mass marker, vitamin B12, in poly-L-lysine (HPLL poly-L-ornithine (HPLO and poly-methylene-co-guanidine hydrochloride (HPMCG membrane alginate microcapsules demonstrate the advantage of using the mass transfer approach to a preliminary screening of various microcapsule formulations. Applying a single marker and evaluating mass transfer coefficients can help to quickly rank the investigated membranes and microcapsules according to their permeability. It has been demonstrated that HPLL, HPLO and HPMCG microcapsules differ from each other by a factor of two concerning the rate of low molecular mass marker transport. Interesting differences in mass transfer through the membrane in both directions in-out was also found, which could possibly be related to the membrane asymmetry.

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

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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.

[The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

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Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

2003-09-01

To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

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Frederick Daidone

Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

The UDP-glucuronate decarboxylase gene family in Populus: structure, expression, and association genetics.

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Qingzhang Du

Full Text Available In woody crop plants, the oligosaccharide components of the cell wall are essential for important traits such as bioenergy content, growth, and structural wood properties. UDP-glucuronate decarboxylase (UXS is a key enzyme in the synthesis of UDP-xylose for the formation of xylans during cell wall biosynthesis. Here, we isolated a multigene family of seven members (PtUXS1-7 encoding UXS from Populus tomentosa, the first investigation of UXSs in a tree species. Analysis of gene structure and phylogeny showed that the PtUXS family could be divided into three groups (PtUXS1/4, PtUXS2/5, and PtUXS3/6/7, consistent with the tissue-specific expression patterns of each PtUXS. We further evaluated the functional consequences of nucleotide polymorphisms in PtUXS1. In total, 243 single-nucleotide polymorphisms (SNPs were identified, with a high frequency of SNPs (1/18 bp and nucleotide diversity (πT = 0.01033, θw = 0.01280. Linkage disequilibrium (LD analysis showed that LD did not extend over the entire gene (r (2<0.1, P<0.001, within 700 bp. SNP- and haplotype-based association analysis showed that nine SNPs (Q <0.10 and 12 haplotypes (P<0.05 were significantly associated with growth and wood property traits in the association population (426 individuals, with 2.70% to 12.37% of the phenotypic variation explained. Four significant single-marker associations (Q <0.10 were validated in a linkage mapping population of 1200 individuals. Also, RNA transcript accumulation varies among genotypic classes of SNP10 was further confirmed in the association population. This is the first comprehensive study of the UXS gene family in woody plants, and lays the foundation for genetic improvements of wood properties and growth in trees using genetic engineering or marker-assisted breeding.

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

DEFF Research Database (Denmark)

Walls, Anne B; Nilsen, Linn Hege; Eyjolfsson, Elvar M

2010-01-01

ABSTRACT: GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission...

Intronic variants in the dopa decarboxylase (DDC) gene are associated with smoking behavior in European-Americans and African-Americans.

Science.gov (United States)

Yu, Yi; Panhuysen, Carolien; Kranzler, Henry R; Hesselbrock, Victor; Rounsaville, Bruce; Weiss, Roger; Brady, Kathleen; Farrer, Lindsay A; Gelernter, Joel

2006-07-15

We report here a study considering association of alleles and haplotypes at the DOPA decarboxylase (DDC) locus with the DSM-IV diagnosis of nicotine dependence (ND) or a quantitative measure for ND using the Fagerstrom Test for Nicotine Dependence (FTND). We genotyped 18 single nucleotide polymorphisms (SNPs) spanning a region of approximately 210 kb that includes DDC and the genes immediately flanking DDC in 1,590 individuals from 621 families of African-American (AA) or European-American (EA) ancestry. Evidence of association (family-based tests) was observed with several SNPs for both traits (0.0002DDC lacking exons 10-15. Haplotype analysis did not reveal any SNP combination with stronger evidence for association than rs12718541 alone. Although sequence analysis suggests that rs12718541 may be an intronic splicing enhancer, further studies are needed to determine whether a direct link exists between an alternatively spliced form of DDC and predisposition to ND. These findings confirm a previous report of association of DDC with ND, localize the causative variants to the 3' end of the coding region and extend the association to multiple population groups.

Analyzing the soybean transcriptome during autoregulation of mycorrhization identifies the transcription factors GmNF-YA1a/b as positive regulators of arbuscular mycorrhization.

Science.gov (United States)

Schaarschmidt, Sara; Gresshoff, Peter M; Hause, Bettina

2013-06-18

Similarly to the legume-rhizobia symbiosis, the arbuscular mycorrhiza interaction is controlled by autoregulation representing a feedback inhibition involving the CLAVATA1-like receptor kinase NARK in shoots. However, little is known about signals and targets down-stream of NARK. To find NARK-related transcriptional changes in mycorrhizal soybean (Glycine max) plants, we analyzed wild-type and two nark mutant lines interacting with the arbuscular mycorrhiza fungus Rhizophagus irregularis. Affymetrix GeneChip analysis of non-inoculated and partially inoculated plants in a split-root system identified genes with potential regulation by arbuscular mycorrhiza or NARK. Most transcriptional changes occur locally during arbuscular mycorrhiza symbiosis and independently of NARK. RT-qPCR analysis verified nine genes as NARK-dependently regulated. Most of them have lower expression in roots or shoots of wild type compared to nark mutants, including genes encoding the receptor kinase GmSIK1, proteins with putative function as ornithine acetyl transferase, and a DEAD box RNA helicase. A predicted annexin named GmAnnx1a is differentially regulated by NARK and arbuscular mycorrhiza in distinct plant organs. Two putative CCAAT-binding transcription factor genes named GmNF-YA1a and GmNF-YA1b are down-regulated NARK-dependently in non-infected roots of mycorrhizal wild-type plants and functional gene analysis confirmed a positive role for these genes in the development of an arbuscular mycorrhiza symbiosis. Our results indicate GmNF-YA1a/b as positive regulators in arbuscular mycorrhiza establishment, whose expression is down-regulated by NARK in the autoregulated root tissue thereby diminishing subsequent infections. Genes regulated independently of arbuscular mycorrhization by NARK support an additional function of NARK in symbioses-independent mechanisms.

A dopa decarboxylase modulating the immune response of scallop Chlamys farreri.

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Zhi Zhou

Full Text Available BACKGROUND: Dopa decarboxylase (DDC is a pyridoxal 5-phosphate (PLP-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. METHODOLOGY: The full-length cDNA encoding DDC (designated CfDDC was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05 at 3 h and reached the peak at 12 h (9.8-fold, P<0.05, and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC was expressed in Escherichia coli BL21 (DE3-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h, and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC. After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05 of blank group at 12 h and 0.47-fold (P<0.05 at 24 h, respectively. CONCLUSIONS: These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.

L-Dopa decarboxylase (DDC) constitutes an emerging biomarker in predicting patients' survival with stomach adenocarcinomas.

Science.gov (United States)

Florou, Dimitra; Papadopoulos, Iordanis N; Fragoulis, Emmanuel G; Scorilas, Andreas

2013-02-01

Stomach adenocarcinoma represents a major health problem and is regarded as the second commonest cause of cancer-associated mortality, universally, since it is still difficult to be perceived at a curable stage. Several lines of evidence have pointed out that the expression of L-Dopa decarboxylase (DDC) gene and/or protein becomes distinctively modulated in several human neuroendocrine neoplasms as well as adenocarcinomas. In order to elucidate the clinical role of DDC on primary gastric adenocarcinomas, we determined qualitatively and quantitatively the mRNA levels of the gene with regular PCR and real-time PCR by using the comparative threshold cycle method, correspondingly, and detected the expression of DDC protein by immunoblotting in cancerous and normal stomach tissue specimens. A statistically significant association was disclosed between DDC expression and gastric intestinal histotype as well as tumor localization at the distal third part of the stomach (p = 0.025 and p = 0.029, respectively). Univariate and multivariate analyses highlighted the powerful prognostic importance of DDC in relation to disease-free survival and overall survival of gastric cancer patients. According to Kaplan-Meier curves, the relative risk of relapse was found to be decreased in DDC-positive (p = 0.031) patients who, also, exhibited higher overall survival rates (p = 0.016) than those with DDC-negative tumors. This work is the first to shed light on the potential clinical usefulness of DDC, as an efficient tumor biomarker in gastric cancer. The provided evidence underlines the propitious predictive value of DDC expression in the survival of stomach adenocarcinoma patients.

Simultaneous detection of diagnostic biomarkers of alkaptonuria, ornithine carbamoyltransferase deficiency, and neuroblastoma disease by high-performance liquid chromatography/tandem mass spectrometry.

Science.gov (United States)

Hsu, Wei-Yi; Chen, Ching-Ming; Tsai, Fuu-Jen; Lai, Chien-Chen

2013-05-01

Urinary homovanillic acid (HVA)/vanillylmandelic acid (VMA), orotic acid (OA), and homogentisic acid (HGA) are diagnostic biomarkers of neuroblastoma, ornithine carbamoyl transferase deficiency (OCTD), and alkaptonuria (AKU), respectively. In this study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of HVA, VMA, OA, and HGA in urine. After sample preparation, which involved only the dilution procedure, samples were quantified by LC-MS/MS. Full-scan MS/MS mode enabled the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC-MS run. The limits of detection were 10 μg/l for HGA, 25 μg/l for HVA/VMA, and 50 μg/l for OA with a single-to-noise ratio of 3; the limits of quantification were 50 μg/l for HVA and HGA, 100 μg/l for VMA, and 250 μg/l for OA. The linear dynamic range for quantification of the analytes covered 2 to 3 orders of magnitude, depending on the analyte. The relative standard deviation of the developed LC-MS/MS method was less than 4% for the intra-day validation and 10% for the inter-day validation. The results show that our LC-MS/MS technique is a highly sensitive and rapid method for screening for biomarkers that are diagnostic of three metabolic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

IGF2BP2 alternative variants associated with glutamic acid decarboxylase antibodies negative diabetes in Malaysian subjects.

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Sameer D Salem

Full Text Available BACKGROUND: The association of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2 common variants (rs4402960 and rs1470579 with type 2 diabetes (T2D has been performed in different populations. The aim of this study was to evaluate the association of alternative variants of IGF2BP2; rs6777038, rs16860234 and rs7651090 with glutamic acid decarboxylase antibodies (GADA negative diabetes in Malaysian Subjects. METHODS/PRINCIPAL FINDINGS: IGF2BP2; rs6777038, rs16860234 and rs7651090 single nucleotide polymorphisms (SNPs were genotyped in 1107 GADA negative diabetic patients and 620 control subjects of Asian from Malaysia. The additive genetic model adjusted for age, race, gender and BMI showed that alternative variants; rs6777038, rs16860234 and rs7651090 of IGF2BP2 associated with GADA negative diabetes (OR = 1.21; 1.36; 1.35, P = 0.03; 0.0004; 0.0002, respectively. In addition, the CCG haplotype and diplotype CCG-TCG increased the risk of diabetes (OR = 1.51, P = 0.01; OR = 2.36, P = 0.009, respectively. CONCLUSIONS/SIGNIFICANCE: IGF2BP2 alternative variants were associated with GADA negative diabetes. The IGF2BP2 haplotypes and diplotypes increased the risk of diabetes in Malaysian subject.

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

International Nuclear Information System (INIS)

Smiley, J.A.; Bell, J.B.; Jones, M.E.; Paneth, P.; O'Leary, M.H.

1991-01-01

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13 C kinetic isotope effect of 1.0247 ± 0.0008 at 25 C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 ± 0.0006 measured at pH 4.0, 25 C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel, which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. These data fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegal

Novel type of ornithine-glutathione double conjugate excreted as a major metabolite into the bile of rats administered clebopride

International Nuclear Information System (INIS)

Ishizuka, T.; Komiya, I.; Hiratsuka, A.; Watabe, T.

1990-01-01

Rats orally given radioactive Clebopride [[14C]CP; N-(1'-benzyl-4'-piperidyl)-2-[14C]methoxy-4-amino-5-chlorobenzamide++ +], an antiulcer agent, excreted a novel type of ornithine (Orn)-GSH double conjugate in the bile as a major metabolite [(14C]BMCP), corresponding to 18% of the dose. The present study provides the first evidence for Orn conjugation of a xenobiotic in mammals and demonstrates that the structure of the radioactive conjugate differs fundamentally from those known in birds and reptiles. The structure of the biliary metabolite, [14C]BMCP, purified to homogeneity by silica gel thin layer and reverse phase high pressure liquid chromatography, was elucidated as S-[2-ornithylamino-4-[14C]methoxy-5-(1'-methyl-4'-piperidylamin o) carboxyphenyl]glutathione, based mainly on the following facts: (1) BMCP showed a protonated molecular ion (M + H)+ peak at m/z 683 in the secondary ion mass spectrum and (2) [14C]BMCP afforded Orn, glutamic acid, glycine, S-(2-amino-4-[14C]methoxy-5-carboxyphenyl)cysteine [( 14C]AMCC), and 1-methyl-4-aminopiperidine (MAP) quantitatively, in an equal molar ratio, by complete hydrolysis with peptidase. Thus, BMCP was a metabolite with three enzymatically hydrolyzable amide bonds in addition to the one existing originally in the parent structure of the drug, which produces MAP by peptic digestion. Of the three additional amide bonds of BMCP, one was a novel type of bond formed by condensation of the alpha-carboxylic acid group of Orn with the primary aromatic amino group of the drug and the other two were in the S-glutathionyl residue, substituted for the chlorine atom vicinal to the Orn-conjugating primary amino group in the aromatic ring and affording glutamic acid, glycine, and the S-cysteine conjugate AMCC by hydrolysis of BMCP with the peptidase

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz

International Nuclear Information System (INIS)

Laier, Peter; Metzdorff, Stine Broeng; Borch, Julie; Hagen, Marie Louise; Hass, Ulla; Christiansen, Sofie; Axelstad, Marta; Kledal, Thuri; Dalgaard, Majken; McKinnell, Chris; Brokken, Leon J.S.; Vinggaard, Anne Marie

2006-01-01

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17α-hydroxylase/17,20-lyase (P450c17) and a reduction in 17β-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level

SNP discovery and High Resolution Melting Analysis from massive transcriptome sequencing in the California red abalone Haliotis rufescens.

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Valenzuela-Muñoz, Valentina; Araya-Garay, José Miguel; Gallardo-Escárate, Cristian

2013-06-01

The California red abalone, Haliotis rufescens that belongs to the Haliotidae family, is the largest species of abalone in the world that has sustained the major fishery and aquaculture production in the USA and Mexico. This native mollusk has not been evaluated or assigned a conservation category even though in the last few decades it was heavily exploited until it disappeared in some areas along the California coast. In Chile, the red abalone was introduced in the 1970s from California wild abalone stocks for the purposes of aquaculture. Considering the number of years that the red abalone has been cultivated in Chile crucial genetic information is scarce and critical issues remain unresolved. This study reports and validates novel single nucleotide polymorphisms (SNP) markers for the red abalone H. rufescens using cDNA pyrosequencing. A total of 622 high quality SNPs were identified in 146 sequences with an estimated frequency of 1 SNP each 1000bp. Forty-five SNPs markers with functional information for gene ontology were selected. Of these, 8 were polymorphic among the individuals screened: Heat shock protein 70 (HSP70), vitellogenin (VTG), lysin, alginate lyase enzyme (AL), Glucose-regulated protein 94 (GRP94), fructose-bisphosphate aldolase (FBA), sulfatase 1A precursor (S1AP) and ornithine decarboxylase antizyme (ODC). Two additional sequences were also identified with polymorphisms but no similarities with known proteins were achieved. To validate the putative SNP markers, High Resolution Melting Analysis (HRMA) was conducted in a wild and hatchery-bred population. Additionally, SNP cross-amplifications were tested in two further native abalone species, Haliotis fulgens and Haliotis corrugata. This study provides novel candidate genes that could be used to evaluate loss of genetic diversity due to hatchery selection or inbreeding effects. Copyright © 2013 Elsevier B.V. All rights reserved.

Regulation of homocysteine metabolism and methylation in human and mouse tissues

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Chen, Natalie C.; Yang, Fan; Capecci, Louis M.; Gu, Ziyu; Schafer, Andrew I.; Durante, William; Yang, Xiao-Feng; Wang, Hong

2010-01-01

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine β-synthase, cystathionine-γ-lyase, paraxonase 1, 5,10-methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyltransferase, phosphatidylethanolamine N-methyltransferases and glycine N-methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl-tRNA synthase, 5-methyltetrahydrofolate:Hcy methyltransferase, S-adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone-lysine N-methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.—Chen, N. C., Yang, F., Capecci, L. M., Gu, Z., Schafer, A. I., Durante, W., Yang, X.-F., Wang, H. Regulation of homocysteine metabolism and methylation in human and mouse tissues. PMID:20305127

Orotidine-5'-monophosphate decarboxylase catalysis: Kinetic isotope effects and the state of hybridization of a bound transition-state analogue

Energy Technology Data Exchange (ETDEWEB)

Acheson, S.A.; Bell, J.B.; Jones, M.E.; Wolfenden, R. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

1990-04-03

The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.

Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

Science.gov (United States)

Goldman, Lawrence M; Amyes, Tina L; Goryanova, Bogdana; Gerlt, John A; Richard, John P

2014-07-16

The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

Effects of temperature, pH and NaCl content on in vitro putrescine and cadaverine production through the growth of Serratia marcescens CCM 303.

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Bubelová, Zuzana; Buňka, František; Taťáková, Monika; Štajnochová, Kateřina; Purevdorj, Khatantuul; Buňková, Leona

2015-01-01

The aim of this study was to evaluate the combined effect of temperature (10, 20 and 37°C), pH (4, 5, 6, 7 and 8), and NaCl content (0, 1, 3, 4, 5 and 6% w/v) on the growth and putrescine and cadaverine production of Serratia marcescens CCM 303 under model conditions. The decarboxylase activity of S. marcescens was monitored in broth after cultivation. The cultivation medium was enriched with selected amino acids (ornithine, arginine and lysine; 0.2% w/v each) serving as precursors of biogenic amines. Levels of putrescine and cadaverine in broth were analysed by high-performance liquid chromatography after pre-column derivatisation with o-phthalaldehyde reagent. S. marcescens produced higher amounts of putrescine (up to 2096.8 mg L(-1)) compared to cadaverine content (up to 343.3 mg L(-1)) in all cultivation media. The highest putrescine and cadaverine concentrations were reached during cultivation at 10-20°C, pH 5-7 and NaCl content 1-3% w/v. On the other hand, the highest BAs production of individual cell (recalculated based on a cell; so called "yield factor") was observed at 10°C, pH 4 and salt concentration 3-5% w/v as a response to environmental stress.

Polyamine deprivation-induced enhanced uptake of methylglyoxal bis(guanylhydrazone) by tumor cells.

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Seppänen, P; Alhonen-Hongisto, L; Jänne, J

1981-05-05

1. Putrescine and spermidine depletion produced by alpha-difluoromethylornithine, an irreversible inhibitor or ornithine decarboxylase (EC 4.1.1.17), resulted in a strikingly enhanced cellular uptake of methylglyoxal bis(guanylhydrazone) in cultured Ehrlich ascites carcinoma cells and human lymphocytic leukemia cells. 2. A prior priming of the cells with difluoromethylornithine followed by a short exposure of the cells to methylglyoxal bis(guanylhydrazone) rapidly established intracellular concentrations of the latter drug approaching 10 mM. 3. The enhanced transport of methylglyoxal bis(guanylhydrazone) into the tumor cells apparently required metabolic energy as the uptake of extracellular drug rapidly ceased and intracellular methylglyoxal bis(guanylhydrazone) was excreted into the medium when the glycolysis of the tumor cells was inhibited by iodoacetate. 4. A sequential treatment of cultured tumor cells with difluoromethylornithine until established polyamine depletion followed by an addition of low concentrations of methylglyoxal bis(guanylhydrazone) produced an antiproliferative action not achieved with either of the drugs alone. 5. A similar treatment schedule, i.e a priming of mice inoculated with Ehrlich ascites cells with difluoromethylornithine for a few days, likewise enhanced the uptake of methylglyoxal bis(guanylhydrazone) by the carcinoma cells, but only marginally increased the drug concentration in the liver and small intestine of the animals.

Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble

DEFF Research Database (Denmark)

Christgau, S; Schierbeck, H; Aanstoot, H J

1991-01-01

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic...... acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular...

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

Science.gov (United States)

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

Long-term dietary restriction up-regulates activity and expression of renal arginase II in aging mice.

Science.gov (United States)

Majaw, T; Sharma, R

2017-06-01

Arginase II is a mitochondrial enzyme that catalyses the hydrolysis of L-arginine into urea and ornithine. It is present in other extra-hepatic tissues that lack urea cycle. Therefore, it is plausible that arginase II has a physiological role other than urea cycle which includes polyamine, proline, glutamate synthesis and regulation of nitric oxide production. The high expression of arginase II in kidney, among extrahepatic tissues, might have an important role associated with kidney functions. The present study is aimed to determine the age-associated alteration in the activity and expression of arginase II in the kidney of mice of different ages. The effect of dietary restriction to modulate the agedependent changes of arginase II was also studied. Results showed that renal arginase II activity declines significantly with the progression of age (p less than 0.01 and p less than 0.001 in 6- and 18-month-old mice, respectively as compared to 2-month old mice) and is due to the reduction in its protein as well as the mRNA level (p less than 0.001 in both 6- and 18-month-old mice as compared to 2-month-old mice). Long-term dietary restriction for three months has significantly up-regulated arginase II activity and expression level in both 2- and 18-month-old mice (p less than 0.01 and p less than 0.001, respectively as compared to AL group). These findings clearly indicate that the reducing level of arginase II during aging might have an impact on the declining renal functions. This age-dependent down-regulation of arginase II in the kidney can be attenuated by dietary restriction which may help in the maintenance of such functions.

Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

Science.gov (United States)

Melo, Nadiele T M; Mulder, Kelly C L; Nicola, André Moraes; Carvalho, Lucas S; Menino, Gisele S; Mulinari, Eduardo; Parachin, Nádia S

2018-02-16

Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris , a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris . To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

In silico screening of potent natural inhibitor compounds against Human DOPA Decarboxylase for management of Parkinson’s Disease

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Surya Narayan Rath

2017-12-01

Full Text Available Loss of dopaminergic neurons of the substantia nigra of the mid brain is a well studied pathophysiology of Parkinson’s disease (PD, is the second most common neurodegenerative disorder. To compensate dopamine levels at the Central Nervous System (CNS exogenous L-Dopa is generally administered. But the major part of the L-Dopa is metabolized by Dopa decarboxylase (DDC, E.C. 4.1.1.28, a pyridoxal 5’ –phosphate (PLP enzyme, which is abundant in CNS and hence, only 1-5% of L-Dopa reaches to dopaminergic neurons. In this context, co-administration of peripheral DDC inhibitors (carbidopa or benserazide has been successfully used for the symptomatic treatment of PD patients. But, due to use of synthetic drugs many adverse effects have been reported during treatment. Therefore, the current study is planned to discover some plant based potent natural inhibitors against human DDC as an alternative way for the management of PD. This study was conducted through virtual screening and molecular docking of DDC enzyme with phytochemicals like withania somnifera (ashwagandha, glycine max (soybean, vicia faba (broad bean, and marsilea quadrifolia (sunsunia etc to evaluate their inhibition properties. In silico study results shown a good binding affinity and predicted some of the phytochemicals as potent natural inhibitors against human DDC. This work could be validated further through experimental procedures.

Malonyl CoA decarboxylase deficiency: C to T transition in intron 2 of the MCD gene.

Science.gov (United States)

Surendran, S; Sacksteder, K A; Gould, S J; Coldwell, J G; Rady, P L; Tyring, S K; Matalon, R

2001-09-15

Malonyl CoA decarboxylase (MCD) is an enzyme involved in the metabolism of fatty acids synthesis. Based on reports of MCD deficiency, this enzyme is particular important in muscle and brain metabolism. Mutations in the MCD gene result in a deficiency of MCD activity, that lead to psychomotor retardation, cardiomyopathy and neonatal death. To date however, only a few patients have been reported with defects in MCD. We report here studies of a patient with MCD deficiency, who presented with hypotonia, cardiomyopathy and psychomotor retardation. DNA sequencing of MCD revealed a homozygous intronic mutation, specifically a -5 C to T transition near the acceptor site for exon 3. RT-PCR amplification of exons 2 and 3 revealed that although mRNA from a normal control sample yielded one major DNA band, the mutant mRNA sample resulted in two distinct DNA fragments. Sequencing of the patient's two RT-PCR products revealed that the larger molecular weight fragments contained exons 2 and 3 as well as the intervening intronic sequence. The smaller size band from the patient contained the properly spliced exons, similar to the normal control. Western blotting analysis of the expressed protein showed only a faint band in the patient sample in contrast to a robust band in the control. In addition, the enzyme activity of the mutant protein was lower than that of the control protein. The data indicate that homozygous mutation in intron 2 disrupt normal splicing of the gene, leading to lower expression of the MCD protein and MCD deficiency. Copyright 2001 Wiley-Liss, Inc.

Nitrogen metabolism, acid-base regulation, and molecular responses to ammonia and acid infusions in the spiny dogfish shark (Squalus acanthias).

Science.gov (United States)

Nawata, C Michele; Walsh, Patrick J; Wood, Chris M

2015-07-01

Although they are ureotelic, marine elasmobranchs express Rh glycoproteins, putative ammonia channels. To address questions raised by a recent study on high environmental ammonia (HEA) exposure, dogfish were intravascularly infused for 24 h at 3 ml kg(-1) h(-1) with isosmotic NaCl (500 mmol l(-1), control), NH4HCO3 (500 mmol l(-1)), NH4Cl (500 mmol l(-1)), or HCl (as 125 mmol l(-1) HCl + 375 mmol l(-1) NaCl). While NaCl had no effect on arterial acid-base status, NH4HCO3 caused mild alkalosis, NH4Cl caused strong acidosis, and HCl caused lesser acidosis, all predominantly metabolic in nature. Total plasma ammonia (T(Amm)) and excretion rates of ammonia (J(Amm)) and urea-N (J(Urea-N)) were unaffected by NaCl or HCl. However, despite equal loading rates, plasma T(Amm) increased to a greater extent with NH4Cl, while J(Amm) increased to a greater extent with NH4HCO3 due to much greater increases in blood-to-water PNH3 gradients. As with HEA, both treatments caused large (90%) elevations of J(Urea-N), indicating that urea-N synthesis by the ornithine-urea cycle (OUC) is driven primarily by ammonia rather than HCO3(-). Branchial mRNA expressions of Rhbg and Rhp2 were unaffected by NH4HCO3 or NH4Cl, but v-type H(+)-ATPase was down-regulated by both treatments, and Rhbg and Na(+)/H(+) exchanger NHE2 were up-regulated by HCl. In the kidney, Rhbg was unresponsive to all treatments, but Rhp2 was up-regulated by HCl, and the urea transporter UT was up-regulated by HCl and NH4Cl. These responses are discussed in the context of current ideas about branchial, renal, and OUC function in this nitrogen-limited predator.

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase.

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Kumar, Vinay; Chattopadhyay, Arnab; Ghosh, Sumit; Irfan, Mohammad; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

2016-06-01

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme. © 2016 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

A Rare Case of Cerebellar Ataxia Due to Voltage-Gated Calcium Channel and Glutamic Acid Decarboxylase Autoantibodies.

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Annunziata, Giuseppe; Lobo, Pamela; Carbuccia, Cristian

2017-11-27

BACKGROUND Autoimmune cerebellar ataxia can be paraneoplastic in nature or can occasionally present without evidence of an ongoing malignancy. The detection of specific autoantibodies has been statistically linked to different etiologies. CASE REPORT A 55-year-old African-American woman with hypertension and a past history of morbid obesity and uncontrolled diabetes status post gastric bypass four years prior to the visit (with significantly improved body mass index and hemoglobin A1c controlled at the time of the clinical encounter) presented to the office complaining of gradual onset of unsteadiness and recurrent falls for the past three years, as well as difficulties coordinating routine daily activities. The neurologic exam showed moderate dysarthria and ataxic gait with bilateral dysmetria and positive Romberg test. Routine laboratory test results were only remarkable for a mild elevation of erythrocyte sedimentation rate, and most laboratory and imaging tests for common causes of ataxia failed to demonstrate an etiology. Upon further workup, evidence of anti-voltage-gated calcium channel and anti-glutamic acid decarboxylase antibody was demonstrated. She was then treated with intravenous immunoglobulins with remarkable clinical improvement. CONCLUSIONS We present a case of antibody-mediated ataxia not associated with malignancy. While ataxia is rarely related to autoantibodies, in such cases it is critical to understand the etiology of this disabling condition in order to treat it correctly. Clinicians should be aware of the possible association with specific autoantibodies and the necessity to rule out an occult malignancy in such cases.

Functional pyruvate formate lyase pathway expressed with two different electron donors in Saccharomyces cerevisiae at aerobic growth

DEFF Research Database (Denmark)

Zhang, Yiming; Dai, Zongjie; Krivoruchko, Anastasia

2015-01-01

pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc-MTH1-ΔT ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression...

Suppression of the formation of polyamines and macromolecules by dl-α-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) in phytohaemagglutinin-activated human lymphocytes

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Jänne, Juhani; Hovi, Tapani; Hölttä, Erkki

1979-01-01

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-α-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1′-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [14C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [3H]uridine and [14C]adenine into total RNA. The

Suppression of the formation of polyamines and macromolecules by DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) in phytohaemagglutinin-activated human lymphocytes.

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Hölttä, E; Jänne, J; Hovi, T

1979-01-15

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA

Carcinogenesis

International Nuclear Information System (INIS)

Reilly, C.A. Jr.

1979-01-01

This section contains summaries of research in the following areas: use of liver for mechanistic studies of multistage hepatocarcinogenesis and for screening of environmental contaminants for tumor initiating and promoting activity; molecular properties of rat liver ornithine aminotransferase; regulation of gene expression in rat liver; methods of tumor detection; mechanisms of radiation and viral oncogenesis; biphenyl metabolism by rat liver microsomes; and studies on aryl hydrocarbon hydroxylase activity

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

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Taupin, P; Ben-Ari, Y; Roisin, M P

1994-05-02

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Systematic study of association of four GABAergic genes: glutamic acid decarboxylase 1 gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor 1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray.

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Zhao, Xu; Qin, Shengying; Shi, Yongyong; Zhang, Aiping; Zhang, Jing; Bian, Li; Wan, Chunling; Feng, Guoyin; Gu, Niufan; Zhang, Guangqi; He, Guang; He, Lin

2007-07-01

Several studies have suggested the dysfunction of the GABAergic system as a risk factor in the pathogenesis of schizophrenia. In the present study, case-control association analysis was conducted in four GABAergic genes: two glutamic acid decarboxylase genes (GAD1 and GAD2), a GABA(A) receptor subunit beta2 gene (GABRB2) and a GABA(B) receptor 1 gene (GABBR1). Using a universal DNA microarray procedure we genotyped a total of 20 SNPs on the above four genes in a study involving 292 patients and 286 controls of Chinese descent. Statistically significant differences were observed in the allelic frequencies of the rs187269C/T polymorphism in the GABRB2 gene (P=0.0450, chi(2)=12.40, OR=1.65) and the -292A/C polymorphism in the GAD1 gene (P=0.0450, chi(2)=14.64 OR=1.77). In addition, using an electrophoretic mobility shift assay (EMSA), we discovered differences in the U251 nuclear protein binding to oligonucleotides representing the -292 SNP on the GAD1 gene, which suggests that the -292C allele has reduced transcription factor binding efficiency compared with the 292A allele. Using the multifactor-dimensionality reduction method (MDR), we found that the interactions among the rs187269C/T polymorphism in the GABRB2 gene, the -243A/G polymorphism in the GAD2 gene and the 27379C/T and 661C/T polymorphisms in the GAD1 gene revealed a significant association with schizophrenia (Pschizophrenia in the Chinese population.

Micro-plate radiobinding assay of autoantibody to glutamic acid decarboxylase

International Nuclear Information System (INIS)

Huang Gan; Jin Helai; Wang Xia; Li Hui; Zhang Song; Zhou Zhiguang

2008-01-01

Objective: The purpose of this study was to develop a high-throughput micro-plate radiobinding assay (RBA) of glutamic acid decarboxylase antibody (CAD-Ab) and to evaluate its clinical application. Methods: 35 S labeled GAD 65 antigen was incubated with sera for 24 h on a 96-well plate, and then transferred to the Millipore plate coated with protein A, which was washed with 4 degree C PBS buffer, and then counted by a liquid scintillation counter. The CAD-Ab results were expressed by WHO standard unit (U/ml). A total of 224 healthy controls, 162 patients with type 1 diabetes mellitus (T1DM) and 210 patients with newly diagnosed type 2 diabetes (T2DM) were recruited. A total of 119 T1DM and healthy eases with gradually changing GAD-Ab levels were selected to compare the consistency of micro-plate RBA with conventional radioligand assay (RLA). Blood samples were obtained from the peripheral vein and finger tip in 32 healthy controls, 35 T1DM and 24 T2DM patients, and tested with micro-plate RBA and then compared with the conventional RLA to investigate the reliability of finger tip sampling. Linear correlation, student's t-test, variance analysis and receiver operating characteristic (ROC) curve were performed using SPSS 11.5. Results: (1) The optimized conditions of micro-plate RBA included 2 μl serum incubated with 3 x 10 4 counts/min 35 S-CAD for 24 h under slow vibration, antigen-antibody compounds washed 10 times by 4 degree C PBS buffer, and radioactivity counted with Optiphase Supermix scintillation liquid. (2)The intra-batch CV of the micro-plate RBA was 3.8%-10.2%, and the inter-batch CV was 5.6%-11.9%. The linearity analysis showed a good correlation when the GAD-Ab in serum samples ranged from 40.3 to 664 U/ml and the detection limit of measurement was 3.6 U/ml. The results from Diabetes Autoantibody Standardization Program (DASP) 2005 showed that the sensitivity and specificity for GAD-Ab were 78% (39 positive among 50 new-onset T1DM) and 98% (2 positive

Markedly Lower Glutamic Acid Decarboxylase 67 Protein Levels in a Subset of Boutons in Schizophrenia.

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Rocco, Brad R; Lewis, David A; Fish, Kenneth N

2016-06-15

Convergent findings indicate that cortical gamma-aminobutyric acid (GABA)ergic circuitry is altered in schizophrenia. Postmortem studies have consistently found lower levels of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) in the prefrontal cortex (PFC) of subjects with schizophrenia. At the cellular level, the density of GABA neurons with detectable levels of GAD67 mRNA is ~30% lower across cortical layers. Knowing how this transcript deficit translates to GAD67 protein levels in axonal boutons is important for understanding the impact it might have on GABA synthesis. In addition, because reductions in GAD67 expression before, but not after, the maturation of GABAergic boutons results in a lower density of GABAergic boutons in mouse cortical cultures, knowing if GABAergic bouton density is altered in schizophrenia would provide insight into the timing of the GAD67 deficit. PFC tissue sections from 20 matched pairs of schizophrenia and comparison subjects were immunolabeled for the vesicular GABA transporter (vGAT) and GAD67. vGAT+ bouton density did not differ between subject groups, consistent with findings that vGAT mRNA levels are unaltered in the illness and confirming that the number of cortical GABAergic boutons is not lower in schizophrenia. In contrast, in schizophrenia subjects, the proportion of vGAT+ boutons with detectable GAD67 levels (vGAT+/GAD67+ boutons) was 16% lower and mean GAD67 levels were 14% lower in the remaining vGAT+/GAD67+ boutons. Our findings suggest that GABA production is markedly reduced in a subset of boutons in the PFC of schizophrenia subjects and that this reduction likely occurs after the maturation of GABAergic boutons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Novel type of ornithine-glutathione double conjugate excreted as a major metabolite into the bile of rats administered clebopride.

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Ishizuka, T; Komiya, I; Hiratsuka, A; Watabe, T

1990-06-01

Rats orally given radioactive Clebopride [[14C]CP; N-(1'-benzyl-4'-piperidyl)-2-[14C]methoxy-4-amino-5-chlorobenzamide++ +], an antiulcer agent, excreted a novel type of ornithine (Orn)-GSH double conjugate in the bile as a major metabolite [( 14C]BMCP), corresponding to 18% of the dose. The present study provides the first evidence for Orn conjugation of a xenobiotic in mammals and demonstrates that the structure of the radioactive conjugate differs fundamentally from those known in birds and reptiles. The structure of the biliary metabolite, [14C]BMCP, purified to homogeneity by silica gel thin layer and reverse phase high pressure liquid chromatography, was elucidated as S-[2-ornithylamino-4-[14C]methoxy-5-(1'-methyl-4'-piperidylamin o) carboxyphenyl]glutathione, based mainly on the following facts: 1) BMCP showed a protonated molecular ion (M + H)+ peak at m/z 683 in the secondary ion mass spectrum and 2) [14C]BMCP afforded Orn, glutamic acid, glycine, S-(2-amino-4-[14C]methoxy-5-carboxyphenyl)cysteine [( 14C]AMCC), and 1-methyl-4-aminopiperidine (MAP) quantitatively, in an equal molar ratio, by complete hydrolysis with peptidase. Thus, BMCP was a metabolite with three enzymatically hydrolyzable amide bonds in addition to the one existing originally in the parent structure of the drug, which produces MAP by peptic digestion. Of the three additional amide bonds of BMCP, one was a novel type of bond formed by condensation of the alpha-carboxylic acid group of Orn with the primary aromatic amino group of the drug and the other two were in the S-glutathionyl residue, substituted for the chlorine atom vicinal to the Orn-conjugating primary amino group in the aromatic ring and affording glutamic acid, glycine, and the S-cysteine conjugate AMCC by hydrolysis of BMCP with the peptidase. Substitution of a methyl group for the benzyl group at the piperidine ring nitrogen atom, leading to the formation of MAP by peptic digestion, also occurred during metabolism of CP to

Chronic periodontitis can affect the levels of potential oral cancer salivary mRNA biomarkers.

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Cheng, Y-S L; Jordan, L; Chen, H-S; Kang, D; Oxford, L; Plemons, J; Parks, H; Rees, T

2017-06-01

More than 100 salivary constituents have been found to show levels significantly different in patients with oral squamous cell carcinoma (OSCC) from those found in healthy controls, and therefore have been suggested to be potential salivary biomarkers for OSCC detection. However, many of these potential OSCC salivary biomarkers are also involved in chronic inflammation, and whether the levels of these biomarkers could be affected by the presence of chronic periodontitis was not known. The objective of this pilot study was therefore to measure the levels of seven previously reported potential OSCC salivary mRNA biomarkers in patients with chronic periodontitis and compare them to levels found in patients with OSCC and healthy controls. The seven salivary mRNAs were interleukin (IL)-8, IL-1β, dual specificity phosphatase 1, H3 histone family 3A, ornithine decarboxylase antizyme 1, S100 calcium-binding protein P (S100P) and spermidine/spermine N1-acetyltransferase 1. Unstimulated whole saliva samples were collected from a total of 105 human subjects from the following four study groups: OSCC; CPNS (chronic periodontitis, moderate to severe degree, non-smokers); CPS (chronic periodontitis, moderate to severe degree, smokers); and healthy controls. Levels of each mRNA in patient groups (OSCC or chronic periodontitis) relative to the healthy controls were determined by a pre-amplification reverse transcription-quantitative polymerase chain reaction approach with nested gene-specific primers. Results were recorded and analyzed by the Bio-Rad CFX96 Real-Time System. Mean fold changes between each pair of patient vs. control groups were analyzed by the Mann-Whitney U-test with Bonferroni corrections. Only S100P showed significantly higher levels in patients with OSCC compared to both patients with CPNS (p = 0.003) and CPS (p = 0.007). The difference in S100P levels between patients with OSCC and healthy controls was also marginally significant (p = 0.009). There was no

Interaction of Human Dopa Decarboxylase with L-Dopa: Spectroscopic and Kinetic Studies as a Function of pH

Directory of Open Access Journals (Sweden)

Riccardo Montioli

2013-01-01

Full Text Available Human Dopa decarboxylase (hDDC, a pyridoxal 5′-phosphate (PLP enzyme, displays maxima at 420 and 335 nm and emits fluorescence at 384 and 504 nm upon excitation at 335 nm and at 504 nm when excited at 420 nm. Absorbance and fluorescence titrations of hDDC-bound coenzyme identify a single pKspec of ~7.2. This pKspec could not represent the ionization of a functional group on the Schiff base but that of an enzymic residue governing the equilibrium between the low- and the high-pH forms of the internal aldimine. During the reaction of hDDC with L-Dopa, monitored by stopped-flow spectrophotometry, a 420 nm band attributed to the 4′-N-protonated external aldimine first appears, and its decrease parallels the emergence of a 390 nm peak, assigned to the 4′-N-unprotonated external aldimine. The pH profile of the spectral change at 390 nm displays a pK of 6.4, a value similar to that (~6.3 observed in both kcat and kcat/Km profiles. This suggests that this pK represents the ESH+ → ES catalytic step. The assignment of the pKs of 7.9 and 8.3 observed on the basic side of kcat and the PLP binding affinity profiles, respectively, is also analyzed and discussed.

Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

OpenAIRE

Driessen, A J; Smid, E J; Konings, W N

1988-01-01

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agma...

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

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Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

2014-05-01

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

Endogenous synthesis of taurine and GABA in rat ocular tissues

Energy Technology Data Exchange (ETDEWEB)

Heinaemaeki, A.A.

1988-01-01

The endogenous production of taurine and ..gamma..-aminobutyric acid (GABA) in rat ocular tissues was investigated. The activities of taurine-producing enzyme, cysteine sulfinic acid decarboxylase (CSAD), and GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), were observed in the retina, lens, iris-ciliary body and cornea. The highest specific activity of CSAD was in the cornea and that of GAD in the retina. The discrepancy between CSAD activity and taurine content within the ocular tissues indicates that intra- or extraocular transport processes may regulate the concentration of taurine on the rat eye. The GAD activity and the content of GABA were distributed in parallel within the rat ocular tissues. The quantitative results suggest that the GAD/GABA system has functional significance only in the retina of the rat eye.

Endogenous synthesis of taurine and GABA in rat ocular tissues

International Nuclear Information System (INIS)

Heinaemaeki, A.A.

1988-01-01

The endogenous production of taurine and γ-aminobutyric acid (GABA) in rat ocular tissues was investigated. The activities of taurine-producing enzyme, cysteine sulfinic acid decarboxylase (CSAD), and GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), were observed in the retina, lens, iris-ciliary body and cornea. The highest specific activity of CSAD was in the cornea and that of GAD in the retina. The discrepancy between CSAD activity and taurine content within the ocular tissues indicates that intra- or extraocular transport processes may regulate the concentration of taurine on the rat eye. The GAD activity and the content of GABA were distributed in parallel within the rat ocular tissues. The quantitative results suggest that the GAD/GABA system has functional significance only in the retina of the rat eye. (author)

Electrochemiluminescence assays for insulin and glutamic acid decarboxylase autoantibodies improve prediction of type 1 diabetes risk.

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Miao, Dongmei; Steck, Andrea K; Zhang, Li; Guyer, K Michelle; Jiang, Ling; Armstrong, Taylor; Muller, Sarah M; Krischer, Jeffrey; Rewers, Marian; Yu, Liping

2015-02-01

We recently developed new electrochemiluminescence (ECL) insulin autoantibody (IAA) and glutamic acid decarboxylase 65 autoantibody (GADA) assays that discriminate high-affinity, high-risk diabetes-specific autoantibodies from low-affinity, low-risk islet autoantibodies (iAbs) detected by radioassay (RAD). Here, we report a further validation of the ECL-IAA and -GADA assays in 3,484 TrialNet study participants. The ECL assay and RAD were congruent in those with prediabetes and in subjects with multiple autoantibodies, but only 24% (P<0.0001) of single RAD-IAA-positive and 46% (P<0.0001) of single RAD-GADA-positive were confirmed by the ECL-IAA and -GADA assays, respectively. During a follow-up (mean, 2.4 years), 51% of RAD-IAA-positive and 63% of RAD-GADA-positive subjects not confirmed by ECL became iAb negative, compared with only 17% of RAD-IAA-positive (P<0.0001) and 15% of RAD-GADA-positive (P<0.0001) subjects confirmed by ECL assays. Among subjects with multiple iAbs, diabetes-free survival was significantly shorter if IAA or GADA was positive by ECL and negative by RAD than if IAA or GADA was negative by ECL and positive by RAD (P<0.019 and P<0.0001, respectively). Both positive and negative predictive values in terms of progression to type 1 diabetes mellitus were superior for ECL-IAA and ECL-GADA, compared with RADs. The prevalence of the high-risk human leukocyte antigen-DR3/4, DQB1*0302 genotype was significantly higher in subjects with RAD-IAA or RAD-GADA confirmed by ECL. In conclusion, both ECL-IAA and -GADA are more disease-specific and better able to predict the risk of progression to type 1 diabetes mellitus than the current standard RADs.

The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

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Dalluge, Joseph J.; Welchlin, Cole W.; Hughes, John; Han, Wei; Blackwell, Timothy S.; Laguna, Theresa A.; Williams, Bryan J.

2014-01-01

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic. PMID:25350753

The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.

Directory of Open Access Journals (Sweden)

Nick B Paulson

Full Text Available The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.

Effect of Lathyrus sativus and vitamin C on the status of aromatic L-amino acid decarboxylase and dipeptidyl-aminopeptidase-IV in the central and peripheral tissues and serum of guinea pigs

International Nuclear Information System (INIS)

Rahman, M.K.; Sarker, M.A.H.

1992-05-01

Studies on the effect of Lathyrus Sativus seeds (LLS) on aromatic L-amino acid decarboxylase (AADC) and on dipeptidyl-aminopeptidase-IV (DAP-IV) were carried out in the central and peripheral tissues and serum of LSS-treated and LSS plus vitamin C-treated guinea pigs. The feeding of LSS for 35 days decreased the AADC activity significantly in the brain and peripheral tissues, but the activity was recovered to normal level in the most tissues when vitamin C was added with the LSS. DAP-IV activity decreased in the peripheral tissues when treated with LSS, but the vitamin C administration with LSS did not recover the enzyme activity. The DAP-IV activity did not decrease significantly in any of the brain tissues of the LSS-treated group. (author). 18 refs, 2 tabs

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

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Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

2015-01-01

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

High titers of autoantibodies to glutamate decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

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Hampe, Christiane S.; Maitland, Murray E.; Gilliam, Lisa K.; Thi Phan, Thanh-H.; Sweet, Ian R.; Radtke, Jared R.; Bota, Vasile; Ransom, Bruce R.; Hirsch, Irl B.

2014-01-01

Objective Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders and patients with type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesize that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. Methods We analyzed GAD65Ab titer, inhibition of GAD65 enzyme activity, and pattern of GAD65Ab epitopes in a cohort of type 1 diabetes patients (n=100) and correlated our findings with neurological symptoms and diseases. Results Fourty-three percent (43/100) of the patients had detectable GAD65Ab titers (median=400 U/ml, range: 142–250,000U/ml). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000–250,000 U/ml). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of five patients inhibited the enzyme activity significantly (by 34–55%). Three of these patients complained of muscle stiffness and pain, which was documented in two of these patients. Conclusions Based on our findings we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. PMID:23512385

Ribosomal protein-Mdm2-p53 pathway coordinates nutrient stress with lipid metabolism by regulating MCD and promoting fatty acid oxidation.

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Liu, Yong; He, Yizhou; Jin, Aiwen; Tikunov, Andrey P; Zhou, Lishi; Tollini, Laura A; Leslie, Patrick; Kim, Tae-Hyung; Li, Lei O; Coleman, Rosalind A; Gu, Zhennan; Chen, Yong Q; Macdonald, Jeffrey M; Graves, Lee M; Zhang, Yanping

2014-06-10

The tumor suppressor p53 has recently been shown to regulate energy metabolism through multiple mechanisms. However, the in vivo signaling pathways related to p53-mediated metabolic regulation remain largely uncharacterized. By using mice bearing a single amino acid substitution at cysteine residue 305 of mouse double minute 2 (Mdm2(C305F)), which renders Mdm2 deficient in binding ribosomal proteins (RPs) RPL11 and RPL5, we show that the RP-Mdm2-p53 signaling pathway is critical for sensing nutrient deprivation and maintaining liver lipid homeostasis. Although the Mdm2(C305F) mutation does not significantly affect growth and development in mice, this mutation promotes fat accumulation under normal feeding conditions and hepatosteatosis under acute fasting conditions. We show that nutrient deprivation inhibits rRNA biosynthesis, increases RP-Mdm2 interaction, and induces p53-mediated transactivation of malonyl-CoA decarboxylase (MCD), which catalyzes the degradation of malonyl-CoA to acetyl-CoA, thus modulating lipid partitioning. Fasted Mdm2(C305F) mice demonstrate attenuated MCD induction and enhanced malonyl-CoA accumulation in addition to decreased oxidative respiration and increased fatty acid accumulation in the liver. Thus, the RP-Mdm2-p53 pathway appears to function as an endogenous sensor responsible for stimulating fatty acid oxidation in response to nutrient depletion.

Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum

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Verónica Gómez Pérez

2016-08-01

Full Text Available Visceral leishmaniasis (VL caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime®, 100 mg/kg/day for 28 days. After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to SbIII for promastigotes and >3-fold to SbIII and 3-fold to SbV for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.

Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease.

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Lowe-Power, Tiffany M; Hendrich, Connor G; von Roepenack-Lahaye, Edda; Li, Bin; Wu, Dousheng; Mitra, Raka; Dalsing, Beth L; Ricca, Patrizia; Naidoo, Jacinth; Cook, David; Jancewicz, Amy; Masson, Patrick; Thomma, Bart; Lahaye, Thomas; Michael, Anthony J; Allen, Caitilyn

2018-04-01

Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

The trophic effect of epidermal growth factor on morphological changes and polyamine metabolism in the small intestine of rats.

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Tsujikawa, T; Bamba, T; Hosoda, S

1990-06-01

This study was undertaken to evaluate the effect of epidermal growth factor (EGF) on the morphological changes and polyamine metabolism in the atrophic small intestinal mucosa of rats caused by feeding elemental diet (ED; Elental, Ajinomoto, Tokyo) for several weeks. Four-week-old Wistar male rats were given ad libitum ED (1 kcal/ml) for 4 weeks. The body weight increased to the same extent as the control group fed a pellet diet. However, the small intestine became atrophic: the mucosal wet weight of the jejunum decreased to 70%, while that of the ileum decreased to 60%. EGF (10 micrograms/kg) was subcutaneously injected into these rats every 8 hours. Ornithine decarboxylase (ODC) activities of the jejunal and ileal mucosa rose within 12 hours of the initial EGF administration. Mucosal DNA specific activities tended to increase. Next, EGF (30 micrograms/kg/day) was intraperitoneally administered with a Mini-osmotic pump for one week. The wet weight, protein and DNA contents of the ileal mucosa increased significantly compared with those of the saline administered controls, while the crypt cell production rate (CCPR) also increased. Histologically, increases in both villus height and crypt depth were confirmed. These findings indicate that EGF causes mucosal proliferation through polyamine metabolism even in the atrophic small intestine of mature rats after ED administration for 4 weeks.

Prevention of short-term ultraviolet B radiation-mediated damages by resveratrol in SKH-1 hairless mice

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Afaq, Farrukh; Adhami, Vaqar Mustafa; Ahmad, Nihal

2003-01-01

Nonmelanoma skin cancer is the most common cancer among humans and solar UV radiation, particularly its UVB component (290-320 nm), is its major cause. One way to reduce the occurrence of the cancer is via the use of substances (often antioxidants) termed 'photochemopreventive agents'. Resveratrol (trans-3,4',5-trihydroxystilbene), a phytoalexin found in grapes, nuts, fruits, and red wine, is a potent antioxidant with strong anti-inflammatory and antiproliferative properties. This study was designed to examine whether resveratrol possesses the potential to ameliorate the damages caused by short-term UVB exposure to mouse skin. Single topical application of resveratrol (25 μmol/0.2 ml acetone per mouse) to SKH-1 hairless mice was found to result in significant inhibition of UVB (180 mJ/cm 2 )-mediated increase in bifold skin thickness and skin edema. The resveratrol treatment to mouse skin was also found to result in significant inhibition of UVB-mediated induction of cyclooxygenase and ornithine decarboxylase (ODC) enzyme activities and protein expression of ODC, which are well-established markers for tumor promotion. We also observed that resveratrol inhibits UVB-mediated increased level of lipid peroxidation, a marker of oxidative stress. Taken together, our results suggest that resveratrol may afford substantial protection against the damages caused by UVB exposure, and these protective effects may be mediated via its antioxidant properties

Biotechnological Applications of Dimorphic Yeasts

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Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

Metabolic regulation of the plant hormone indole-3-acetic acid

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Jerry D. Cohen

2009-11-01

The phytohormone indole-3-acetic acid (IAA, auxin) is important for many aspects of plant growth, development and responses to the environment yet the routes to is biosynthesis and mechanisms for regulation of IAA levels remain important research questions. A critical issue concerning the biosynthesis if IAA in plants is that redundant pathways for IAA biosynthesis exist in plants. We showed that these redundant pathways and their relative contribution to net IAA production are under both developmental and environmental control. We worked on three fundamental problems related to how plants get their IAA: 1) An in vitro biochemical approach was used to define the tryptophan dependent pathway to IAA using maize endosperm, where relatively large amounts of IAA are produced over a short developmental period. Both a stable isotope dilution and a protein MS approach were used to identify intermediates and enzymes in the reactions. 2) We developed an in vitro system for analysis of tryptophan-independent IAA biosynthesis in maize seedlings and we used a metabolite profiling approach to isolate intermediates in this reaction. 3) Arabidopsis contains a small family of genes that encode potential indolepyruvate decarboxylase enzymes. We cloned these genes and studied plants that are mutant in these genes and that over-express each member in the family in terms of the level and route of IAA biosynthesis. Together, these allowed further development of a comprehensive picture of the pathways and regulatory components that are involved in IAA homeostasis in higher plants.

Ca2+ and aminoguanidine on γ-aminobutyric acid accumulation in germinating soybean under hypoxia–NaCl stress

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Runqiang Yang

2015-06-01

Full Text Available Gamma-aminobutyric acid (GABA, a nonproteinous amino acid with some benefits on human health, is synthesized by GABA-shunt and the polyamine degradation pathway in plants. The regulation of Ca2+ and aminoguanidine on GABA accumulation in germinating soybean (Glycine max L. under hypoxia-NaCl stress was investigated in this study. Exogenous Ca2+ increased GABA content significantly by enhancing glutamate decarboxylase gene expression and its activity. Addition of ethylene glycol tetra-acetic acid into the culture solution reduced GABA content greatly due to the inhibition of glutamate decarboxylase activity. Aminoguanidine reduced over 85% of diamine oxidase activity, and 33.28% and 36.35% of GABA content in cotyledon and embryo, respectively. Under hypoxia–NaCl stress, the polyamine degradation pathway contributed 31.61–39.43% of the GABA formation in germinating soybean.

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

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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. Copyright © 2015 Elsevier B.V. All rights reserved.

Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features.

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Curley, Allison A; Arion, Dominique; Volk, David W; Asafu-Adjei, Josephine K; Sampson, Allan R; Fish, Kenneth N; Lewis, David A

2011-09-01

Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia. GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.

Prevalence and Clinical Characteristics of Recently Diagnosed Type 2 Diabetes Patients with Positive Anti-Glutamic Acid Decarboxylase Antibody

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Yul Hwangbo

2012-04-01

Full Text Available BackgroundLatent autoimmune diabetes in adults (LADA refers to a specific type of diabetes characterized by adult onset, presence of islet auto-antibodies, insulin independence at the time of diagnosis, and rapid decline in β-cell function. The prevalence of LADA among patients with type 2 diabetes varies from 2% to 20% according to the study population. Since most studies on the prevalence of LADA performed in Korea were conducted in patients who had been tested for anti-glutamic acid decarboxylase antibody (GADAb, a selection bias could not be excluded. In this study, we examined the prevalence and clinical characteristics of LADA among adult patients recently diagnosed with type 2 diabetes.MethodsWe included 462 patients who were diagnosed with type 2 diabetes within 5 years from the time this study was performed. We measured GADAb, fasting insulin level, fasting C-peptide level, fasting plasma glucose level, HbA1c, and serum lipid profiles and collected data on clinical characteristics.ResultsThe prevalence of LADA was 4.3% (20/462 among adult patients with newly diagnosed type 2 diabetes. Compared with the GADAb-negative patients, the GADAb-positive patients had lower fasting C-peptide levels (1.2±0.8 ng/mL vs. 2.0±1.2 ng/mL, P=0.004. Other metabolic features were not significantly different between the two groups.ConclusionThe prevalence of LADA is 4.3% among Korean adult patients with recently diagnosed type 2 diabetes. The Korean LADA patients exhibited decreased insulin secretory capacity as reflected by lower C-peptide levels.

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65

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Maxwell Denis

2008-11-01

Full Text Available Abstract Background Human glutamic acid decarboxylase 65 (hGAD65 is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution. Results A DNA cassette encoding full-length hGAD65, under the control of the C. reinhardtii chloroplast rbcL promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of C. reinhardtii by particle bombardment. Integration of hGAD65 DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of hGAD65 was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein. Conclusion Our results demonstrate the potential value of C. reinhardtii chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.

Transplastomic expression of bacterial L-aspartate-alpha-decarboxylase enhances photosynthesis and biomass production in response to high temperature stress.

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Fouad, W M; Altpeter, F

2009-10-01

Metabolic engineering for beta-alanine over-production in plants is expected to enhance environmental stress tolerance. The Escherichia coli L-aspartate-alpha-decarboxylase (AspDC) encoded by the panD gene, catalyzes the decarboxylation of L-aspartate to generate beta-alanine and carbon dioxide. The constitutive E. coli panD expression cassette was co-introduced with the constitutive, selectable aadA expression cassette into the chloroplast genome of tobacco via biolistic gene transfer and homologous recombination. Site specific integration of the E. coli panD expression cassette into the chloroplast genome and generation of homotransplastomic plants were confirmed by PCR and Southern blot analysis, respectively, following plant regeneration and germination of seedlings on selective media. PanD expression was verified by assays based on transcript detection and in vitro enzyme activity. The AspDC activities in transplastomic plants expressing panD were drastically increased by high-temperature stress. beta-Alanine accumulated in transplastomic plants at levels four times higher than in wildtype plants. Analysis of chlorophyll fluorescence on plants subjected to severe heat stress at 45 degrees C under light verified that photosystem II (PSII) in transgenic plants had higher thermotolerance than in wildtype plants. The CO(2) assimilation of transplastomic plants expressing panD was more tolerant to high temperature stress than that of wildtype plants, resulting in the production of 30-40% more above ground biomass than wildtype control. The results presented indicate that chloroplast engineering of the beta-alanine pathway by over-expression of the E. coli panD enhances thermotolerance of photosynthesis and biomass production following high temperature stress.

[Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata].

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Peng, Silu; Yang, Huilin; Zhu, Du; Zhang, Zhibin; Yan, Riming; Wang, Ya

2016-04-14

Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET- 22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24 °C, 200 r/min with 1×10–3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni2+ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

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Linsalata, Michele; Giannini, Romina; Notarnicola, Maria; Cavallini, Aldo

2006-01-01

The peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N 1 -acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARγ expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARγ expression were also investigated in terms of K-ras mutation. PPARγ, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC). ODC and SSAT activity were measured by a radiometric technique. PPARγ expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARγ and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p < 0.0001; r = 0.65, p < 0.0001, respectively). Moreover, gene expression, polyamine levels and enzymatic activities were increased in colorectal carcinoma samples expressing K-ras mutation as compared to non mutated K-ras samples. In conclusion, our data demonstrated a close relationship between PPARγ and SSAT in human colorectal cancer and this could represent an attempt to decrease polyamine levels and to reduce cell

Glutamic acid decarboxylase-derived epitopes with specific domains expand CD4(+CD25(+ regulatory T cells.

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Guojiang Chen

Full Text Available BACKGROUND: CD4(+CD25(+ regulatory T cell (Treg-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. METHODOLOGY/PRINCIPAL FINDINGS: Splenic lymphocytes from nonobese diabetic (NOD mice were stimulated with different glutamic acid decarboxylase (GAD-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538-expanded CD4(+CD25(+ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528- or p530 (GAD530-543-expanded CD4(+CD25(+ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes between the above-mentioned epitopes and MHC class II I-A(g7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7. Furthermore, p524 bound to I-A(g7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570, which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+CD25(+ T cells suppressed the onset of diabetes in NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of

Tyrosine Hydroxylase (TH)- and Aromatic-L-Amino Acid Decarboxylase (AADC)-Immunoreactive Neurons of the Common Marmoset (Callithrix jacchus) Brain: An Immunohistochemical Analysis

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Karasawa, Nobuyuki; Hayashi, Motoharu; Yamada, Keiki; Nagatsu, Ikuko; Iwasa, Mineo; Takeuchi, Terumi; Uematsu, Mitsutoshi; Watanabe, Kazuko; Onozuka, Minoru

2007-01-01

From the perspective of comparative morphology, the distribution of non-monoaminergic neurons in the common marmoset (Callithrix jacchus) was investigated using an immunohistochemical method with specific antibodies to tyrosine hydroxylase (TH) and aromatic-L-amino acid decarboxylase (AADC). TH-immunoreactive (IR) neurons (but not AADC-IR) neurons were observed in the olfactory tubercle, preoptic suprachiasmatic nucleus, periventricular hypothalamic nucleus, arcuate nucleus, paraventricular nucleus, periaqueductal gray matter, medial longitudinal fasciculus, substantia nigra, and nucleus solitaris. In contrast, AADC-IR (but not TH-IR), small, oval and spindle-shaped neurons were sparsely distributed in the following areas: the hypothalamus from the anterior nucleus to the lateral nucleus, the dorsomedial nucleus, the dorsomedial area of the medial mammillary nucleus and the arcuate nucleus; the midbrain, including the stria medullaris and substantia nigra; and the medulla oblongata, including the dorsal area of the nucleus solitaris and the medullary reticular nucleus. The distribution of AADC-IR neurons was not as extensive in the marmoset as it is in rats. However, these neurons were located in the marmoset, but not the rat substantia nigra. Furthermore, AADC-IR neurons that are present in the human striatum were absent in that of the marmoset. The present results indicate that the distribution of non-monoaminergic neurons in the brain of the common marmoset is unique and different from that in humans and rodents. PMID:17653300