Sample records for acid decarboxylase gad

  1. Alterations in cerebellar glutamic acid decarboxylase (GAD) activity in a genetic model of torsion dystonia (rat). (United States)

    Oltmans, G A; Beales, M; Lorden, J F; Gordon, J H


    Glutamic acid decarboxylase (GAD) activity was studied in specific brain regions of a newly identified genetic (rat) model of human torsion dystonia. GAD activity was found to be significantly increased in the deep cerebellar nuclei of dystonic rats at 16, 20, and 24 days of age. GAD activity in the other regions examined (vermis, cerebellar hemispheres, caudate nucleus, and globus pallidus) did not differ from that of age-matched normal littermate controls. Diazepam treatment significantly reduced the frequency of dystonic movements in the mutant.

  2. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases. (United States)

    Monnerie, Hubert; Le Roux, Peter D


    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

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

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


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

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

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


    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.

  5. MDMA decreases glutamic acid decarboxylase (GAD) 67-immunoreactive neurons in the hippocampus and increases seizure susceptibility: Role for glutamate. (United States)

    Huff, Courtney L; Morano, Rachel L; Herman, James P; Yamamoto, Bryan K; Gudelsky, Gary A


    3,4-Methylenedioxy-methamphetamine (MDMA) is a unique psychostimulant that continues to be a popular drug of abuse. It has been well documented that MDMA reduces markers of 5-HT axon terminals in rodents, as well as humans. A loss of parvalbumin-immunoreactive (IR) interneurons in the hippocampus following MDMA treatment has only been documented recently. In the present study, we tested the hypothesis that MDMA reduces glutamic acid decarboxylase (GAD) 67-IR, another biochemical marker of GABA neurons, in the hippocampus and that this reduction in GAD67-IR neurons and an accompanying increase in seizure susceptibility involve glutamate receptor activation. Repeated exposure to MDMA (3×10mg/kg, ip) resulted in a reduction of 37-58% of GAD67-IR cells in the dentate gyrus (DG), CA1, and CA3 regions, as well as an increased susceptibility to kainic acid-induced seizures, both of which persisted for at least 30days following MDMA treatment. Administration of the NMDA antagonist MK-801 or the glutamate transporter type 1 (GLT-1) inducer ceftriaxone prevented both the MDMA-induced loss of GAD67-IR neurons and the increased vulnerability to kainic acid-induced seizures. The MDMA-induced increase in the extracellular concentration of glutamate in the hippocampus was significantly diminished in rats treated with ceftriaxone, thereby implicating a glutamatergic mechanism in the neuroprotective effects of ceftriaxone. In summary, the present findings support a role for increased extracellular glutamate and NMDA receptor activation in the MDMA-induced loss of hippocampal GAD67-IR neurons and the subsequent increased susceptibility to evoked seizures.

  6. Assessment of CD4+ T cell responses to glutamic acid decarboxylase 65 using DQ8 tetramers reveals a pathogenic role of GAD65 121-140 and GAD65 250-266 in T1D development.

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    I-Ting Chow

    Full Text Available Susceptibility to type 1 diabetes (T1D is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02. Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65 using DQ8 tetramers. We demonstrated that GAD65 121-140 and GAD65 250-266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65 121-140 and GAD65 250-266 carried a Th1-dominant phenotype, with some of the GAD65 121-140-specific T cell clones producing IL-17. GAD65 250-266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs revealed that GAD65 250-266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65 121-140 and GAD65 250-266 epitopes and implicate their possible contribution to the progression of T1D.

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

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


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

  8. Epigenetic signature of panic disorder: a role of glutamate decarboxylase 1 (GAD1) DNA hypomethylation? (United States)

    Domschke, Katharina; Tidow, Nicola; Schrempf, Marie; Schwarte, Kathrin; Klauke, Benedikt; Reif, Andreas; Kersting, Anette; Arolt, Volker; Zwanzger, Peter; Deckert, Jürgen


    Glutamate decarboxylases (GAD67/65; GAD1/GAD2) are crucially involved in gamma-aminobutyric acid (GABA) synthesis and thus were repeatedly suggested to play an important role in the pathogenesis of anxiety disorders. In the present study, DNA methylation patterns in the GAD1 and GAD2 promoter and GAD1 intron 2 regions were investigated for association with panic disorder, with particular attention to possible effects of environmental factors. Sixty-five patients with panic disorder (f=44, m=21) and 65 matched healthy controls were analyzed for DNA methylation status at 38 GAD1 promoter/intron2 and 10 GAD2 promoter CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. Recent positive and negative life events were ascertained. Patients and controls were genotyped for GAD1 rs3762556, rs3791878 and rs3762555, all of which are located in the analyzed promoter region. Patients with panic disorder exhibited significantly lower average GAD1 methylation than healthy controls (p<0.001), particularly at three CpG sites in the promoter as well as in intron 2. The occurrence of negative life events was correlated with relatively decreased average methylation mainly in the female subsample (p=0.01). GAD1 SNP rs3762555 conferred a significantly lower methylation at three GAD1 intron 2 CpG sites (p<0.001). No differential methylation was observed in the GAD2 gene. The present pilot data suggest a potentially compensatory role of GAD1 gene hypomethylation in panic disorder possibly mediating the influence of negative life events and depending on genetic variation. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

  9. Identification and transcript analysis of two glutamate decarboxylase genes, CsGAD1 and CsGAD2, reveal the strong relationship between CsGAD1 and citrate utilization in citrus fruit. (United States)

    Liu, Xiao; Hu, Xiao-Mei; Jin, Long-Fei; Shi, Cai-Yun; Liu, Yong-Zhong; Peng, Shu-Ang


    Glutamate decarboxylase (GAD, EC has been suggested to be a key, regulatory point in the biosynthesis of γ-aminobutyrate (GABA) and in the utilization of citric acid through GABA shunt pathway. In this study we discovered two GAD genes, named as CsGAD1 and CsGAD2, in citrus genome database and then successfully cloned. Both CsGAD1 and CsGAD2 have a putative pyridoxal 5-phosphate binding domain in the middle region and a putative calmodulin-binding domain at the carboxyl terminus. Gene structure analysis showed that much difference exists in the size of exons and introns or in cis-regulatory elements in promoter region between the two GAD genes. Gene expression indicated that CsGAD1 transcript was predominantly expressed in flower and CsGAD2 transcript was predominantly expressed in fruit juice sacs; in the ripening fruit, CsGAD1 transcript level was at least 2-time higher than CsGAD2 transcript level. Moreover, CsGAD1 transcript level was increased significantly along with the increase of GAD activity and accompanied by a significant decrease of titratable acid (TA), suggesting that it is CsGAD1 rather than CsGAD2 plays a role in the citric acid utilization during fruit ripening. In addition, injection of abscisic acid and foliar spray of K2SO4 significantly increased the TA content of Satsuma mandarin, and significantly decreased GAD activity as well as CsGAD1 transcript, further suggesting the important role of CsGAD1 in the citrate utilization of citrus fruit.

  10. The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis. (United States)

    Bouché, Nicolas; Fait, Aaron; Zik, Moriyah; Fromm, Hillel


    In plants, as in most eukaryotes, glutamate decarboxylase catalyses the synthesis of GABA. The Arabidopsis genome contains five glutamate decarboxylase genes and one of these genes (glutamate decarboxylase1; i.e. GAD1 ) is expressed specifically in roots. By isolating and analyzing three gad1 T-DNA insertion alleles, derived from two ecotypes, we investigated the potential role of GAD1 in GABA production. We also analyzed a promoter region of the GAD1 gene and show that it confers root-specific expression when fused to reporter genes. Phenotypic analysis of the gad1 insertion mutants revealed that GABA levels in roots were drastically reduced compared with those in the wild type. The roots of the wild type contained about sevenfold more GABA than roots of the mutants. Disruption of the GAD1 gene also prevented the accumulation of GABA in roots in response to heat stress. Our results show that the root-specific calcium/calmodulin-regulated GAD1 plays a major role in GABA synthesis in plants under normal growth conditions and in response to stress.

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

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


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

  12. Cerebellar Ataxia and Glutamic Acid Decarboxylase Antibodies (United States)

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


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

  13. Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders. (United States)

    Pearce, David A; Atkinson, Mark; Tagle, Danilo A


    Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

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

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


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

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

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    Giancarlo eRusso


    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.

  16. Gender differences in associations of glutamate decarboxylase 1 gene (GAD1 variants with panic disorder.

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    Heike Weber

    Full Text Available BACKGROUND: Panic disorder is common (5% prevalence and females are twice as likely to be affected as males. The heritable component of panic disorder is estimated at 48%. Glutamic acid dehydrogenase GAD1, the key enzyme for the synthesis of the inhibitory and anxiolytic neurotransmitter GABA, is supposed to influence various mental disorders, including mood and anxiety disorders. In a recent association study in depression, which is highly comorbid with panic disorder, GAD1 risk allele associations were restricted to females. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen single nucleotide polymorphisms (SNPs tagging the common variation in GAD1 were genotyped in two independent gender and age matched case-control samples (discovery sample n = 478; replication sample n = 584. Thirteen SNPs passed quality control and were examined for gender-specific enrichment of risk alleles associated with panic disorder by using logistic regression including a genotype×gender interaction term. The latter was found to be nominally significant for four SNPs (rs1978340, rs3762555, rs3749034, rs2241165 in the discovery sample; of note, the respective minor/risk alleles were associated with panic disorder only in females. These findings were not confirmed in the replication sample; however, the genotype×gender interaction of rs3749034 remained significant in the combined sample. Furthermore, this polymorphism showed a nominally significant association with the Agoraphobic Cognitions Questionnaire sum score. CONCLUSIONS/SIGNIFICANCE: The present study represents the first systematic evaluation of gender-specific enrichment of risk alleles of the common SNP variation in the panic disorder candidate gene GAD1. Our tentative results provide a possible explanation for the higher susceptibility of females to panic disorder.

  17. Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis

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    Haixiong Liu; Shifeng Li; Yunbin Zhang; Yuanchang Yan; Yiping Li


    Glutamate decarboxylase 65 (GAD65) produces γ-amino-butyric acid,the main inhibitory neurotransmitter in adult mammalian brain.Previous experiments,per-formed in brain,showed that GAD65 gene possesses two TATA-less promoters,although the significance is unknown.Here,by rapid amplification of cDNA ends method,two distinct GAD65 mRNA isoforms transcribed from two independent clusters of transcription start sites were identified in post-natal rat testis.RT-PCR results revealed that the two mRNA isoforms had distinct expression patterns during post-natal testis maturation,suggesting that GAD65 gene expression was regulated by alternative promoters at the transcription level.By using GAD65-speciflc antibodies,western blotting analysis showed that the 58-kDa GAD65,N-terminal 69 amino acids truncated form of full-length GAD65 protein,was developmentally expressed during post-natal testis matu-ration,suggesting that GAD65 gene expression in testis may also be regulated by post-translational processing.Confocal immunofluorescence microscopy revealed that GAD65 protein was presented in Leydig cells of Day 1 testis,primary spermatocytes and spermatids of post-natal of Day 90 testis.The above results suggested that GAD65 gene expression is dynamically regulated at mul-tiple levels during post-natal testis maturation.

  18. Apraxia in anti-glutamic acid decarboxylase-associated stiff person syndrome: link to corticobasal degeneration? (United States)

    Bowen, Lauren N; Subramony, S H; Heilman, Kenneth M


    Corticobasal syndrome (CBS) is associated with asymmetrical rigidity as well as asymmetrical limb-kinetic and ideomotor apraxia. Stiff person syndrome (SPS) is characterized by muscle stiffness and gait difficulties. Whereas patients with CBS have several forms of pathology, many patients with SPS have glutamic acid decarboxylase antibodies (GAD-ab), but these 2 disorders have not been reported to coexist. We report 2 patients with GAD-ab-positive SPS who also had signs suggestive of CBS, including asymmetrical limb rigidity associated with both asymmetrical limb-kinetic and ideomotor apraxia. Future studies should evaluate patients with CBS for GAD-ab and people with SPS for signs of CBS.

  19. 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;


    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....

  20. Gluconic acid production by gad mutant of Klebsiella pneumoniae. (United States)

    Wang, Dexin; Wang, Chenhong; Wei, Dong; Shi, Jiping; Kim, Chul Ho; Jiang, Biao; Han, Zengsheng; Hao, Jian


    Klebsiella pneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1 g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1 g/g.

  1. 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.

  2. Immunotherapy-responsive limbic encephalitis with antibodies to glutamic acid decarboxylase. (United States)

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


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

  3. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast

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    Agarwal Pankaj


    Full Text Available Stiff limb syndrome (SLS is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, baclofen and steroids.This is the third reported case of SLS as a paraneoplastic accompaniment to cancer.

  4. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast



    Stiff limb syndrome (SLS) is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab) are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, ba...

  5. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast. (United States)

    Agarwal, Pankaj A; Ichaporia, Nasli R


    Stiff limb syndrome (SLS) is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab) are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, baclofen and steroids.This is the third reported case of SLS as a paraneoplastic accompaniment to cancer.

  6. Characteristic expression of γ-aminobutyric acid and glutamate decarboxylase in rat jejunum and its relation to differentiation of epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Fang-Yu Wang; Masahito Watanabe; Ren-Min Zhu; Kentaro Maemura


    AIM: To investigate the expression between γ-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum.METHODS: Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms,GAD65 and GAD67) was investigated in rat jejunum.Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore,evaluation of cell kinetics in jejunum was conducted by 3Hthymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA).RESULTS: The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells.3H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone.CONCLUSION: The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.

  7. Cloning and molecular evolution research of porcine GAD65 gene

    Institute of Scientific and Technical Information of China (English)

    YU Hao; SONG Yuefen; LI Li; LIU Di


    Glutamate decarboxylase (GAD) has been found in animal and higher plant tissues as well as in yeasts and microorganisms.In animals the enzyme plays an important role in central nervous system activity because the enzyme substrate glutamic acid is a mediator of excitation process and the product, gamma-aminobutyric acid, is the most important mediator of inhibition process in the central nervous system. GAD65 is one form of the glutamate decarboxylases (GAD), GAD65 has been identified as a major autoantigen in type 1 diabetes, so the GAD65 gene of porcine was cloned by RT-PCR method to construct phylogenetic tree, the homology of 13glutamate decarboxylases (GAD) of different origin was analyzed by multiple alignment.

  8. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions. (United States)

    Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra


    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.

  9. Developmental changes of glutamate acid decarboxylase 67 in mouse brain after hypoxia ischemia

    Institute of Scientific and Technical Information of China (English)

    Fa-Lin XU; Chang-Lian ZHU; Xiao-Yang WANG


    Objective To study the developmental changes of glutamic acid decarboxylase-67 ( GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6 ±7.0)%TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.

  10. Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Márcio S Baptista

    Full Text Available Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during

  11. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus (United States)

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

  12. Characterization of striatal neurons expressing high levels of glutamic acid decarboxylase messenger RNA. (United States)

    Chesselet, M F; Robbins, E


    Two types of labelled cells are detected in sections of rat and mouse striata processed for in situ hybridization histochemistry with 35S-radiolabelled RNA probes complementary to the messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD), the synthesis enzyme for gamma-aminobutyric acid (GABA): numerous lightly, and fewer very densely labelled neurons. In order to determine whether the densely labelled cells correspond to the striatal somatostatinergic neurons with which they share morphological characteristics, the presence of GAD mRNA was examined in brain sections processed successively for dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, a marker of striatal somatostatinergic neurons, and in situ hybridization histochemistry. In addition, the distribution of GABAergic interneurons was analyzed with regard to striatal compartments (striosomes) indicated by patches of dense opiate binding sites. The results show that NADPH diaphorase activity and GAD mRNA do not co-exist in striatal neurons. Furthermore, in contrast to the somatostatinergic neurons which are almost exclusively located in the extrastriosomal matrix, densely labelled GAD cells were present both in the striosomes and the matrix, further suggesting that GABAergic and somatostatinergic neurons form two distinct interneuronal systems in the striatum of rats and mice.

  13. Glutamic acid decarboxylase autoantibody-positivity post-partum is associated with impaired β-cell function in women with gestational diabetes mellitus

    DEFF Research Database (Denmark)

    Lundberg, T. P.; Højlund, K.; Snogdal, L. S.;


    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...

  14. Sequential elevation of autoantibodies to thyroglobulin and glutamic acid decarboxylase in type 1 diabetes

    Institute of Scientific and Technical Information of China (English)

    Eiji; Kawasaki; Jun-ichi; Yasui; Masako; Tsurumaru; Haruko; Takashima; Toshiyuki; Ikeoka; Fumi; Mori; Satoru; Akazawa; Ikuko; Ueki; Masakazu; Kobayashi; Hironaga; Kuwahara; Norio; Abiru; Hironori; Yamasaki; Atsushi; Kawakami


    We have previously reported the high levels of glutamic acid decarboxylase 65 autoantibodies(GAD65A)in patients with type 1 diabetes and autoimmune thyroid disease.Here we describe a 32-year-old Japanese female with a thirteen-year history of type 1 diabetes whose levels of GAD65A were elevated just after the emergence of anti-thyroid autoimmunity.At 19 years of age,she developed diabetic ketoacidosis and was diagnosed with type 1 diabetes.She had GAD65A,insulinoma-associated antigen-2 autoantibodies(IA-2A),and zinc transporter-8 autoantibodies(ZnT8A),but was negative for antibodies to thyroid peroxidase(TPOAb)and thyroglobulin(TGAb)at disease onset.ZnT8A and IA-2A turned negative 2-3 years after the onset,whereas GAD65A were persistently positive at lower level(approximately 40 U/mL).However,just after the emergence of TGAb at disease duration of 12.5 years,GAD65A levels were reelevated up to5717 U/mL in the absence of ZnT8A and IA-2A.Her thyroid function was normal and TPOAb were consistently negative.She has a HLA-DRB1*03:01/*04:01-DQB1*02:01/*03:02 genotype.Persistent positivity for GAD65A might be associated with increased risk to develop anti-thyroid autoimmunity.

  15. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production. (United States)

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


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

  16. Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. (United States)

    Herbison, A E; Augood, S J; McGowan, E M


    Evidence suggests that medial preoptic area (MPOA) neurones containing gamma-aminobutyric acid (GABA) are modulated directly by oestrogen. We have used an alkaline phosphatase-labelled antisense oligonucleotide probe to examine glutamic acid decarboxylase67 (GAD) mRNA expression within individual cells of the MPOA, diagonal band of Broca (DBB) and parietal cortex in rats killed at noon on each day of the oestrous cycle and after ovariectomy (n = 4-5). As a fall in extracellular GABA concentrations occurs in the MPOA on the afternoon of proestrus, the GAD67 mRNA content of cells was also examined in proestrous rats at 15:00h immediately prior to the preovulatory luteinising hormone (LH) surge. The MPOA was found to have an intermediate number of GAD67 mRNA-containing cells compared with the DBB and cortex (P less than 0.01) but expressed the lowest mean hybridisation signal (P less than 0.01). The parietal cortex had significantly fewer (P less than 0.01) GAD mRNA-containing cells than either the MPOA or DBB but these contained higher mean density of signal (P less than 0.01). The hybridisation signal for GAD mRNA was abolished by either ribonuclease pre-treatment or the use of excess non-labelled probe. No significant (P greater than 0.05) differences in GAD67 mRNA were detected in animals killed at noon throughout the oestrous cycle or after ovariectomy. On the afternoon of proestrus (15:00h) there was a significant 40% reduction in mean GAD67 mRNA content within cells of only the MPOA compared with noon (P less than 0.05). The numbers of cells in the MPOA expressing GAD67 mRNA were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat. (United States)

    Caboche, Jocelyne; Vernier, Philippe; Rogard, Monique; Julien, Jean-François; Mallet, Jacques; Besson, Marie-Jo


    Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also

  18. GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders. (United States)

    Fouka, P; Alexopoulos, H; Akrivou, S; Trohatou, O; Politis, P K; Dalakas, M C


    Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GAD-specific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes.

  19. Glutamic acid decarboxylase-67-positive hippocampal interneurons undergo a permanent reduction in number following kainic acid-induced degeneration of ca3 pyramidal neurons. (United States)

    Shetty, A K; Turner, D A


    Kainic acid (KA)-induced degeneration of CA3 pyramidal neurons leads to synaptic reorganization and hyperexcitability in both dentate gyrus and CA1 region of the hippocampus. We hypothesize that the substrate for hippocampal inhibitory circuitry incurs significant and permanent alterations following degeneration of CA3 pyramidal neurons. We quantified changes in interneuron density (N(v)) in all strata of the dentate gyrus and the CA1 and CA3 subfields of adult rats at 1, 4, and 6 months following intracerebroventricular (icv) KA administration, using glutamic acid decarboxylase-67 (GAD-67) immunocytochemistry. At 1 month postlesion, GAD-67-positive interneuron density was significantly reduced in all strata of every hippocampal region except stratum pyramidale of CA1. The reduction in GAD-67-positive interneuron density either persisted or exacerbated at 4 and 6 months postlesion in every stratum of all hippocampal regions. Further, the soma of remaining GAD-67-positive interneurons in dentate gyrus and CA3 subfield showed significant hypertrophy. Thus, both permanent reductions in the density of GAD-67-positive interneurons in all hippocampal regions and somatic hypertrophy of remaining GAD-67-positive interneurons in dentate gyrus and CA3 subfield occur following icv KA. In contrast, the density of interneurons visualized with Nissl in CA1 and CA3 regions was nearly equivalent to that in the intact hippocampus at all postlesion time points. Collectively, these results suggest that persistent reductions in GAD-67-positive interneuron density observed throughout the hippocampus following CA3 lesion are largely due to a permanent loss of GAD-67 expression in a significant fraction of interneurons, rather than widespread degeneration of interneurons. Nevertheless, a persistent decrease in interneuron activity, as evidenced by permanent down-regulation of GAD-67 in a major fraction of interneurons, would likely enhance the degree of hyperexcitability in the CA3

  20. Anti-Glutamic Acid Decarboxylase Antibody-Associated Ataxia as an Extrahepatic Autoimmune Manifestation of Hepatitis C Infection: A Case Report

    Directory of Open Access Journals (Sweden)

    Amer Awad


    Full Text Available Extrahepatic immunological manifestations of hepatitis C virus (HCV are well described. In addition, antiglutamic acid decarboxylase (GAD antibody-associated cerebellar ataxia is well-established entity. However, there have been no reports in the literature of anti-GAD antibody-associated ataxia as an extrahepatic manifestation of HCV infection. We report the case of a young woman with chronic hepatitis C virus and multiple extrahepatic autoimmune diseases including Sjögren syndrome and pernicious anemia who presented with subacute midline cerebellar syndrome and was found to have positive antiglutamic acid decarboxylase (GAD antibody in the serum and cerebrospinal fluid. An extensive diagnostic workup to rule out neoplastic growths was negative, suggesting the diagnosis of nonparaneoplastic antiglutamic acid decarboxylase antibody-associated cerebellar ataxia as an additional extrahepatic manifestation of hepatitis C virus infection. The patient failed to respond to high-dose steroids and intravenous immunoglobulin. Treatment with the monoclonal antibody rituximab stabilized the disease. We postulate that anti-GAD associated ataxia could be an extrahepatic manifestation of HCV infection.

  1. Anti-glutamic Acid decarboxylase antibody-associated ataxia as an extrahepatic autoimmune manifestation of hepatitis C infection: a case report. (United States)

    Awad, Amer; Stüve, Olaf; Mayo, Marlyn; Alkawadri, Rafeed; Estephan, Bachir


    Extrahepatic immunological manifestations of hepatitis C virus (HCV) are well described. In addition, antiglutamic acid decarboxylase (GAD) antibody-associated cerebellar ataxia is well-established entity. However, there have been no reports in the literature of anti-GAD antibody-associated ataxia as an extrahepatic manifestation of HCV infection. We report the case of a young woman with chronic hepatitis C virus and multiple extrahepatic autoimmune diseases including Sjögren syndrome and pernicious anemia who presented with subacute midline cerebellar syndrome and was found to have positive antiglutamic acid decarboxylase (GAD) antibody in the serum and cerebrospinal fluid. An extensive diagnostic workup to rule out neoplastic growths was negative, suggesting the diagnosis of nonparaneoplastic antiglutamic acid decarboxylase antibody-associated cerebellar ataxia as an additional extrahepatic manifestation of hepatitis C virus infection. The patient failed to respond to high-dose steroids and intravenous immunoglobulin. Treatment with the monoclonal antibody rituximab stabilized the disease. We postulate that anti-GAD associated ataxia could be an extrahepatic manifestation of HCV infection.

  2. Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain. (United States)

    Najlerahim, A; Harrison, P J; Barton, A J; Heffernan, J; Pearson, R C


    In situ hybridization histochemistry (ISHH) using synthetic oligonucleotide probes has been used to identify cells containing the mRNAs coding for glutaminase (GluT), aspartate aminotransferase (AspT) and glutamic acid decarboxylase (GAD). The distribution of GAD mRNA confirms previous descriptions and matches the distribution of GAD detected using specific antibodies. AspT mRNA is widely distributed in the brain, but is present at high levels in GABAergic neuronal populations, some that may be glutamatergic, and in a subset of neurons which do not contain significant levels of either GAD or GluT mRNA. Particularly prominent are the neurons of the magnocellular division of the red nucleus, the large cells in the deep cerebellar nuclei and the vestibular nuclei and neurons of the lateral superior olivary nucleus. GluT mRNA does not appear to be present at high levels in all GAD-containing neurons, but is seen prominently in many neuronal populations that may use glutamate as a neurotransmitter, such as neocortical and hippocampal pyramidal cells, the granule cells of the cerebellum and neurons of the dentate gyrus of the hippocampus. The heaviest labelling of GluT mRNA is seen in the lateral reticular nucleus of the medulla. ISHH using probes directed against the mRNAs encoding these enzymes may be an important technique for identifying glutamate and aspartate using neuronal populations and for examining their regulation in a variety of experimental and pathological circumstances.

  3. Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons.

    Directory of Open Access Journals (Sweden)

    Jamil Kanaani

    Full Text Available The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

  4. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

    DEFF Research Database (Denmark)

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.;


    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehens...

  5. Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain. (United States)

    Ferraguti, F; Zoli, M; Aronsson, M; Agnati, L F; Goldstein, M; Filer, D; Fuxe, K


    The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number

  6. Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli. (United States)

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


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

  7. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism. (United States)

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


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

  8. Glutamic acid decarboxylase 65: a link between GABAergic synaptic plasticity in the lateral amygdala and conditioned fear generalization. (United States)

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


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

  9. Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli. (United States)

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


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

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

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


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

  11. Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat. (United States)

    Yuan, P Q; Grånäs, C; Källström, L; Yu, J; Huhman, K; Larhammar, D; Albers, H E; Johnson, A E


    The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the

  12. Relationship between serum GAD-Ab and the genetic polymorphisms of GAD2 and type 2 diabetes mellitus. (United States)

    Li, Q; Qiao, Z R; Liu, D B; Zeng, J T; Zhang, J; Bo, Y; Zu, H Y; Hu, Q; Wu, X; Dong, S S


    In this study, we investigated the relationship between serum glutamic acid decarboxylase (GAD) autoantibody (Ab) levels and single nucleotide polymorphisms (SNPs) of the glutamic acid de-carboxylase 2 (GAD2) 5'-untranslated region and the susceptibility to type 2 diabetes in the Han population. The distributions of patients with SNPs in the GAD2 5'-untranslated region (rs2236418, rs185649317, and rs8190590) and type 2 diabetes and that of the healthy group were genotyped and analyzed using Sequenom MassArray SNP genotyp-ing. GAD-Ab levels were also detected. The frequency distributions of the AA, AG, and GG genotypes in the polymorphic site rs2236418 in the diabetes GAD-Ab-positive group were 45.9, 42.8, and 11.4%, respectively, whereas those in the control group were 36.6, 43.7, and 19.8%, respectively. The difference between the 2 groups was statis-tically significant (P 2.623 (1.351-4.937) and 2.152 (1.375-4.202), respectively). The associations of the 3 SNPs of the GAD2 gene 5'-un-translated region polymorphisms with susceptibility to type 2 diabe-tes in the Chongqing Han population were significant. The SNP of rs2236418 in the Chongqing Han population of diabetic patients with serum GAD-Ab levels was significantly correlated with the SNPs rs185649317 and rs8190590.

  13. Elimination of islet cell antibodies and glutamic acid decarboxylase antibodies II in a patient with newly diagnosed insulin-dependent diabetes mellitus. (United States)

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


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

  14. Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons. (United States)

    Stone, D M; Grillo, M; Margolis, F L; Joh, T H; Baker, H


    Expression of the dopaminergic phenotype in olfactory bulb (OB) juxtaglomerular neurons (constituting a population of periglomerular and external tufted cells) is dependent upon functional innervation by peripheral olfactory receptors. Loss of functional input in rodents, by either peripheral deafferentation or deprivation of odorant access, results in a profound decrease in the expression of juxtaglomerular tyrosine hydroxylase (TH). We have examined the effects of such treatments on the expression of the neurotransmitter biosynthetic enzyme glutamic acid decarboxylase (GAD), which is colocalized with TH in the majority of TH-containing juxtaglomerular neurons. Following either chemically induced OB deafferentation in adult mice or unilateral odor deprivation in neonatal rats, steady-state OB GAD messenger RNA levels remained essentially unchanged as assessed by Northern blot analysis 20-40 days after treatment. These results were confirmed by in situ hybridization analysis, which demonstrated a profound loss of juxtaglomerular TH messenger RNA but no accompanying decrease in regionally colocalized GAD message. Since GAD is found in nearly all dopaminergic OB cells, the preservation of juxtaglomerular GAD message implies that olfactory receptor neurons exert a differential transneuronal regulation of TH and GAD gene transcription.

  15. Role of dopamine in the plasticity of glutamic acid decarboxylase messenger RNA in the rat frontal cortex and the nucleus accumbens. (United States)

    Rétaux, S; Trovero, F; Besson, M J


    The modulatory role of dopamine (DA) on the expression of mRNA encoding the large isoform of glutamic acid decarboxylase (GAD67), the biosynthesis enzyme of gamma aminobutyric acid (GABA), was examined in GABA neurons of two structures innervated by DA neurons originating from the ventral tegmental area (VTA): the medial frontal cortex (MFC) and the nucleus accumbens (NAcc). A bilateral electrolytic lesion of VTA was performed in rats to produce a DA denervation of both the MFC and NAcc. The efficacy of VTA lesions was verified by measurement of locomotor activity and by immunohistochemical detection of tyrosine hydroxylase in the mesencephalon. GAD67 mRNA was detected by in situ hybridization histochemistry using a 35S-labelled cDNA probe. Densitometric analysis of GAD67 mRNA hybridization signals revealed in VTA-lesioned rats a significant decrease (-24%) in GAD67 mRNA levels in the prelimbic area of the MFC and no significant effect in the anterior cingulate area or the frontoparietal cortex. Single cell analyses by computer-assisted grain counting showed that the decrease in GAD67 mRNA levels in prelimbic MFC was due to a change in GAD67 mRNA expression in a subpopulation of GABA interneurons located in the deep cortical layers (V-VI). By contrast, in the NAcc of VTA-lesioned rats, GAD67 mRNA levels were significantly increased in the anterior part and in the core but were unchanged in the shell part. These results suggest that in two target structures of VTA DA neurons, GAD67 mRNA expression is, in normal conditions, under a tonic stimulatory and a tonic inhibitory DA control in the MFC and the NAcc respectively. A schematic diagram is proposed for functional interactions between these structures.

  16. 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;


    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...

  17. Stiff Person syndrome and other anti-GAD-associated neurologic disorders. (United States)

    Dayalu, Praveen; Teener, James W


    Antibodies directed against glutamic acid decarboxylase (GAD) are present in many patients with stiff person syndrome and increasingly found in patients with other symptoms indicative of central nervous system (CNS) dysfunction, such as ataxia. The classic clinical features of stiff person syndrome include muscular stiffness with superimposed painful muscular spasms. Gait is often impaired. Other CNS disorders associated with GAD antibodies include progressive encephalomyelitis with rigidity and myoclonus (PERM), limbic encephalitis, and even epilepsy. Glutamic acid decarboxylase is the rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter. Presumably, antibodies directed against GAD impair GABA production, but the precise pathogenic mechanism of GAD-antibody-related neurologic disorders is uncertain. Many patients respond to treatment with immunomodulating therapy. Symptomatic treatment with agents that enhance GABA activity, such as benzodiazepines and baclofen, is also helpful for many patients.

  18. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals. (United States)

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


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

  19. Adult and embryonic GAD transcripts are spatiotemporally regulated during postnatal development in the rat brain.

    Directory of Open Access Journals (Sweden)

    Anke Popp

    Full Text Available BACKGROUND: GABA (gamma-aminobutyric acid, the main inhibitory neurotransmitter in the brain, is synthesized by glutamic acid decarboxylase (GAD. GAD exists in two adult isoforms, GAD65 and GAD67. During embryonic brain development at least two additional transcripts exist, I-80 and I-86, which are distinguished by insertions of 80 or 86 bp into GAD67 mRNA, respectively. Though it was described that embryonic GAD67 transcripts are not detectable during adulthood there are evidences suggesting re-expression under certain pathological conditions in the adult brain. In the present study we systematically analyzed for the first time the spatiotemporal distribution of different GADs with emphasis on embryonic GAD67 mRNAs in the postnatal brain using highly sensitive methods. METHODOLOGY/PRINCIPAL FINDINGS: QPCR was used to precisely investigate the postnatal expression level of GAD related mRNAs in cortex, hippocampus, cerebellum, and olfactory bulb of rats from P1 throughout adulthood. Within the first three postnatal weeks the expression of both GAD65 and GAD67 mRNAs reached adult levels in hippocampus, cortex, and cerebellum. The olfactory bulb showed by far the highest expression of GAD65 as well as GAD67 transcripts. Embryonic GAD67 splice variants were still detectable at birth. They continuously declined to barely detectable levels during postnatal development in all investigated regions with exception of a comparatively high expression in the olfactory bulb. Radioactive in situ hybridizations confirmed the occurrence of embryonic GAD67 transcripts in the olfactory bulb and furthermore detected their localization mainly in the subventricular zone and the rostral migratory stream. CONCLUSIONS/SIGNIFICANCE: Embryonic GAD67 transcripts can hardly be detected in the adult brain, except for specific regions associated with neurogenesis and high synaptic plasticity. Therefore a functional role in processes like proliferation, migration or

  20. Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis. (United States)

    Kass, Itamar; Hoke, David E; Costa, Mauricio G S; Reboul, Cyril F; Porebski, Benjamin T; Cowieson, Nathan P; Leh, Hervé; Pennacchietti, Eugenia; McCoey, Julia; Kleifeld, Oded; Borri Voltattorni, Carla; Langley, David; Roome, Brendan; Mackay, Ian R; Christ, Daniel; Perahia, David; Buckle, Malcolm; Paiardini, Alessandro; De Biase, Daniela; Buckle, Ashley M


    The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

  1. The expanding spectrum of pediatric anti-glutamic acid decarboxylase antibody mediated CNS disease - a chance association?

    Institute of Scientific and Technical Information of China (English)

    Deepak Menon; Ramshekhar N Menon; Hardeep Kumar; Ashalatha Radhakrishnan; Sudheeran Kannoth; Muralidharan Nair; Sanjeev Thomas


    Central nervous system autoimmunity in the pediatric age group represents an evolving constellation of various syndromes distinct from the adult age group. One of the rarely described pathogenic auto-antibodies (ab) is the one directed against glutamic acid decarboxylase (GAD). While its pathogenic role is controversial, literature concerning adult patients abounds with heterogeneous presentations with epilepsy often as part of limbic encephalitis or chronic temporal lobe epilepsy and cerebellar ataxia accompanying endocrinopathies or paraneoplastic disorders. Diagnosis is often delayed until late adulthood. The authors report hitherto under-reported syndromes in the pediatric age group. The ifrst case was a 3-year-old boy with sub-acute myoclonus-ataxia following a lfu-like illness akin to para-infectious cerebellitis. The second case was a 7-year-old girl with long-standing chronic extratemporal partial epilepsy and electrical status epilepticus in sleep (ESES) with right hemiparesis and developmental delay. Investigations revealed two-four fold elevations in titres of GAD-65-ab. The absence of systemic diseases like diabetes and the dramatic clinical response to steroids as well as intravenous immunoglobulin in both the cases argued for GAD-ab mediated neuronal injury rather than a chance association. The concern exists regarding other potentially co-existent auto-ab to gamma-amino butyric acid and glycine receptors, and demonstration of intrathecal synthesis of GAD-ab would be ideal. This entity should be contemplated in children presenting with acute/sub-acute onset episodic or progressive ataxia or refractory cryptogenic focal epilepsy syndromes, epileptic encephalopathy such as ESES and worsening neurological deifcits. These children ought to be maintained on regular follow-up for monitoring evolution of other autoimmune disorders in adult life.

  2. Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution. (United States)

    Zik, M; Arazi, T; Snedden, W A; Fromm, H


    The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

  3. Progressive loss of glutamic acid decarboxylase, parvalbumin, and calbindin D28K immunoreactive neurons in the cerebral cortex and hippocampus of adult rat with experimental hydrocephalus. (United States)

    Tashiro, Y; Chakrabortty, S; Drake, J M; Hattori, T


    The authors investigated functional neuronal changes in experimental hydrocephalus using immunohistochemical techniques for glutamic acid decarboxylase (GAD) and two neuronal calcium-binding proteins: parvalbumin (PV) and calbindin D28K (CaBP). Hydrocephalus was induced in 16 adult Wistar rats by intracisternal injection of a kaolin solution, which was confirmed microscopically via atlantooccipital dural puncture. Four control rats received the same volume of sterile saline. Immunohistochemical staining for GAD, PV, and CaBP, and Nissl staining were performed at 1, 2, 3, and 4 weeks after the injection. Hydrocephalus occurred in 90% of kaolin-injected animals with various degrees of ventricular dilation. In the cerebral cortex, GAD-, PV-, and CaBP-immunoreactive (IR) interneurons initially lost their stained processes together with a concomitant loss of homogeneous neuropil staining, followed by the reduction of their total number. With progressive ventricular dilation, GAD- and PV-IR axon terminals on the cortical pyramidal cells disappeared, whereas the number of CaBP-IR pyramidal cells decreased, and ultimately in the most severe cases of hydrocephalus, GAD, PV, and CaBP immunoreactivity were almost entirely diminished. In the hippocampus, GAD-, PV-, and CaBP-IR interneurons demonstrated a reduction of their processes and terminals surrounding the pyramidal cells, with secondary reduction of CaBP-IR pyramidal and granular cells. On the other hand, Nissl staining revealed almost no morphological changes induced by ischemia or neuronal degeneration even in the most severe cases of hydrocephalus. Hydrocephalus results in the progressive functional impairment of GAD-, PV-, and CaBP-IR neuronal systems in the cerebral cortex and hippocampus, often before there is evidence of morphological injury. The initial injury of cortical and hippocampal interneurons suggests that the functional deafferentation from intrinsic projection fibers may be the initial neuronal event

  4. [Inhibitory effect of essential oils, food additives, peracetic acid and detergents on bacterial histidine decarboxylase]. (United States)

    Kamii, Eri; Terada, Gaku; Akiyama, Jyunki; Isshiki, Kenji


    The aim of this study is to examine whether various essential oils, food additives, peracetic acid and detergents inhibit bacterial histidine decarboxylase. Crude extract of Morganella morganii NBRC3848 was prepared and incubated with various agents. Histidine decarboxylase activity was significantly inhibited (pperacetic acid caused slight decomposition. Histidine and histamine were stable in the presence of the other 24 agents. These results indicated that 25 of the agents examined were inhibitors of histidine decarboxylase.

  5. Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice. (United States)

    Alonso-Espinaco, V; Elezgarai, I; Díez-García, J; Puente, N; Knöpfel, T; Grandes, P


    Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.

  6. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study. (United States)

    Verimli, Ural; Sehirli, Umit S


    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.

  7. 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. (United States)

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


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

  8. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase. (United States)

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


    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.

  9. Consequence of nigrostriatal denervation and L-dopa therapy on the expression of glutamic acid decarboxylase messenger RNA in the pallidum. (United States)

    Herrero, M T; Levy, R; Ruberg, M; Luquin, M R; Villares, J; Guillen, J; Faucheux, B; Javoy-Agid, F; Guridi, J; Agid, Y; Obeso, J A; Hirsch, E C


    To examine the consequences of nigrostriatal denervation and L-dopa treatment on the basal ganglia output system, we analyzed, by quantitative in situ hybridization, the messenger RNA coding for glutamic acid decarboxylase (Mr 67,000) (GAD67 mRNA) in pallidal cells from patients with Parkinson's disease (PD), monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) receiving or not receiving L-dopa, and their respective control subjects. In MPTP-treated monkeys, the expression of GAD67 mRNA was increased in cells from the internal pallidum, and this effect was abolished by L-dopa treatment. There were no differences in the levels of GAD67 mRNA between patients with PD, who were all treated with L-dopa, and control subjects. These results indicate that the level of GAD67 mRNA is increased in the cells of the internal pallidum after nigrostriatal dopaminergic denervation and that this increase can be reversed by L-dopa therapy.

  10. Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons

    DEFF Research Database (Denmark)

    Kanaani, Jamil; Cianciaruso, Chiara; Phelps, Edward A;


    The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of...

  11. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase. (United States)

    Dougherty, Charles M; Dayan, Jean


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

  12. GAD65 autoantibodies and its role as biomarker of Type 1 diabetes and Latent Autoimmune Diabetes in Adults (LADA)


    Towns, Roberto; Pietropaolo, Massimo


    One of the hallmarks of autoimmune diabetes is the presence of adaptive responses directed to neuroendocrine proteins. One of these proteins is glutamic acid decarboxylase (GAD). While GAD is widely distributed in neuroendocrine tissues, its specific significance in diabetes has paralleled the advances in understanding humoral and cellular immunity in Type 1 diabetes (T1D) and in a subset of Type 2 diabetes (T2D), going from the seminal discoveries of islet autoantibodies to the development a...

  13. Detection and transfer of the glutamate decarboxylase gene in Streptococcus thermophilus (United States)

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

  14. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110 (United States)

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

  15. GAD2 Alternative Transcripts in the Human Prefrontal Cortex, and in Schizophrenia and Affective Disorders.

    Directory of Open Access Journals (Sweden)

    Kasey N Davis

    Full Text Available Genetic variation and early adverse environmental events work together to increase risk for schizophrenia. γ-aminobutyric acid (GABA, the major inhibitory neurotransmitter in adult mammalian brain, plays a major role in normal brain development, and has been strongly implicated in the pathobiology of schizophrenia. GABA synthesis is controlled by two glutamic acid decarboxylase (GAD genes, GAD1 and GAD2, both of which produce a number of alternative transcripts. Genetic variants in the GAD1 gene are associated with increased risk for schizophrenia, and reduced expression of its major transcript in the human dorsolateral prefrontal cortex (DLPFC. No consistent changes in GAD2 expression have been found in brains from patients with schizophrenia. In this work, with the use of RNA sequencing and PCR technologies, we confirmed and tracked the expression of an alternative truncated transcript of GAD2 (ENST00000428517 in human control DLPFC homogenates across lifespan besides the well-known full length transcript of GAD2. In addition, using quantitative RT-PCR, expression of GAD2 full length and truncated transcripts were measured in the DLPFC of patients with schizophrenia, bipolar disorder and major depression. The expression of GAD2 full length transcript is decreased in the DLPFC of schizophrenia and bipolar disorder patients, while GAD2 truncated transcript is increased in bipolar disorder patients but decreased in schizophrenia patients. Moreover, the patients with schizophrenia with completed suicide or positive nicotine exposure showed significantly higher expression of GAD2 full length transcript. Alternative transcripts of GAD2 may be important in the growth and development of GABA-synthesizing neurons as well as abnormal GABA signaling in the DLPFC of patients with schizophrenia and affective disorders.

  16. Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

    NARCIS (Netherlands)

    Pennacchietti, E.; Lammens, T.M.; Capitani, G.; Franssen, M.C.R.; John, R.A.; Bossa, F.; Biase, De D.


    Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5'-phosphate-dependent enzyme catalyzing CO2 release from the a-carboxyl group of l-glutamate to yield ¿-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperat

  17. GAD67 deficiency in parvalbumin interneurons produces deficits in inhibitory transmission and network disinhibition in mouse prefrontal cortex. (United States)

    Lazarus, Matthew S; Krishnan, Keerthi; Huang, Z Josh


    In mammalian neocortex, the delicate balance of neural circuits is regulated by a rich repertoire of inhibitory control mechanisms mediated by diverse classes of GABAergic interneurons. A key step common to all GABAergic neurons is the synthesis of GABA, catalyzed by 2 isoforms of glutamic acid decarboxylases (GAD). Among these, GAD67 is the rate-limiting enzyme. GAD67 level is regulated by neural activity and is altered in multiple neuropsychiatric disorders. The significance of altered GAD67 levels on inhibitory transmission, however, remains unclear. The presence of GAD65, postsynaptic GABA receptor regulation, and the diversity of cortical interneurons make the link from GAD67 levels to GABA transmission less than straightforward. Here, we selectively removed one allele of the GAD67 gene, Gad1, in PV interneurons in juvenile mice. We found substantial deficits in transmission from PV to pyramidal neurons in prefrontal cortex, along with increases of pyramidal cell excitability and excitation/inhibition balance in PV cells. Synaptic deficits recovered in adult mice, suggesting engagement of homeostatic and compensatory mechanisms. These results demonstrate that GAD67 levels directly influence synaptic inhibition. Thus, GAD67 deficiency in PV cells likely contributes to cortical dysfunction in disease states; the reversibility of synaptic deficits suggests nonpermanent damage to inhibitory circuitry.

  18. Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH (United States)

    Wu, Qinglong; Tun, Hein Min; Law, Yee-Song; Khafipour, Ehsan; Shah, Nagendra P.


    Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis. PMID:28261168

  19. 谷氨酸脱羧酶抗体在2型糖尿病患者中的检出率及分布特征%Analysis of the Detection Rate and Distrubution Feature of Glutamic Acid Decarboxylase Antibodies in Patients with Type 2 Diabetes Mellitus

    Institute of Scientific and Technical Information of China (English)

    张利方; 刘莹; 石莉萍; 汪薇


    Objective: To explore the change of detection rate and distribution feature of glutamic acid decarboxylase antibody (GAD-Ab) in patients with type 2 diabetes mellitus (T2DM). Method: To detect the positive rate of GAD-Ab using EL1SA method in 1970 patients with T2DM and its distribution in different sex, age, course of disease and body mass index (BM1). Fasting, postprandial insulin 2H (FINS, 2hLNS) and fasting, postprandial 2h C peptide (FCP, 2hCP) level were detected by electro-chemiluminescence in GAD-Ab positive and negative patients and compared with the healthy people. Results: 126 cases were GAD-Ab positive among the 1970 patients with T2DM and the total positive rate was 6. 39%. GAD-Ab was mainly distributed in patients during the 30 ~49 years old (P0.05).GAD-Ab阳性组T2DM FINS、2hINS、FCP和2hCP均低于GAD-Ab阴性组和健康对照组(P<0.05).结论:GAD-Ab检测有助于临床对T2DM患者胰岛素功能变化进行评价.

  20. Evolution and expression analysis of the soybean glutamate decarboxylase gene family

    Indian Academy of Sciences (India)

    Tae Kyung Hyun; Seung Hee Eom; Xiao Han; Ju-Sung Kim


    Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate into -aminobutyric acid (GABA), which is a four-carbon non-protein amino acid present in all organisms. Although plant GAD plays important roles in GABA biosynthesis, our knowledge concerning GAD gene family members and their evolutionary relationship remains limited. Therefore, in this study, we have analysed the evolutionary mechanisms of soybean GAD genes and suggested that these genes expanded in the soybean genome partly due to segmental duplication events. The approximate dates of duplication events were calculated using the synonymous substitution rate, and we suggested that the segmental duplication of GAD genes in soybean originated 9.47 to 11.84 million years ago (Mya). In addition, all segmental duplication pairs (GmGAD1/3 and GmGAD2/4) are subject to purifying selection. Furthermore, GmGAD genes displayed differential expression either in their transcript abundance or in their expression patterns under abiotic stress conditions like salt, drought, and cold. The expression pattern of paralogous pairs suggested that they might have undergone neofunctionalization during the subsequent evolution process. Taken together, our results provide valuable information for the evolution of the GAD gene family and represent the basis for future research on the functional characterization of GAD genes in higher plants.

  1. Characterization of hydroxyl radical modified GAD65: a potential autoantigen in type 1 diabetes. (United States)

    Khan, Mohd Wajid A; Sherwani, Subuhi; Khan, Wahid A; Ali, Rashid


    Glutamic acid decarboxylase-65 (GAD(65)) is an immunological marker of type 1 autoimmune diabetes. High titre of autoantibodies against GAD(65) (GAD(65)Abs) have also been detected in some other autoimmune diseases. In search of a potential immunological marker of type 1 diabetes, in vitro GAD(65) was modified by hydroxyl radical followed by the study of structural and conformational perturbed protein by different spectroscopic techniques (UV, fluorescence and CD) and thermal denaturation profile. Binding studies of circulating autoantibodies from diabetic groups (type 1 and type 2) with native and reactive oxygen species (ROS) modified GAD(65), exhibited high recognition of type 1 diabetic serum autoantibodies with modified antigen (p group and control group (n = 10) was checked by inhibition enzyme linked immunosorbent assay (ELISA) and quantitative precipitin titration assay. Relative affinity of ROS-GAD(65)Abs for modified and native GAD(65) was in the order of 1.56 x 10(- 6) and 2.72 x 10(- 7) M, as calculated by Langmuir plot. In coherence, ROS oxidation of GAD(65) causes conformational perturbation, generating highly immunogenic unique neoepitopes that may be one of the factors in antigen-driven induction of type 1 diabetes autoantibodies that can serve as a potential marker in early diagnosis/prognosis of the disease.

  2. Immunology of stiff person syndrome and other GAD-associated neurological disorders. (United States)

    Alexopoulos, Harry; Dalakas, Marinos C


    Antibodies against glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of GABA, are associated with an array of distinct, mostly autoimmune, neurological conditions. In all associated syndromes, namely stiff person syndrome, cerebellar ataxia, epilepsy, limbic encephalitis or abnormal eye movements, anti-GAD antibodies are detected at high titers and play a fundamental role in diagnosis, but do not correlate with disease severity, diversity of symptomatology or response to therapies. Despite considerable efforts, including in vitro (enzymatic assays) and in vivo (animal models) systems, the pathogenicity of anti-GAD antibodies has not been unequivocally proven for any specific condition. The search for the responsible autoantigen has revealed a few other antigenic targets, particularly for SPS, localized in the pre- or post-synaptic inhibitory neuronal synapses. Cumulative clinical and laboratory evidence indicates that anti-GAD and related antibodies define a novel group of syndromes, collectively known as 'hyperexcitability disorders'.

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

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


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

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

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


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

  5. Sexually dimorphic expression of glutamate decarboxylase mRNA in the hypothalamus of the deep sea armed grenadier, Coryphaenoides (Nematonurus) armatus. (United States)

    Trudeau, V L; Bosma, P T; Collins, M; Priede, I G; Docherty, K


    Glutamate decarboxylase (GAD), is a key enzyme in the central nervous system (CNS) that synthesizes the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) from glutamate. Our previous phylogenetic studies on the evolution of this enzyme indicates that there are at least two distinct forms: GAD65 and GAD67. They are the products of separate genes and probably derive from a common ancestral GAD gene following gene duplication prior to the emergence of the teleosts more than 200 Myr ago. Furthermore, a third GAD-like molecule, GAD3, discovered in the armed grenadier, Coryphaenoides (Nematonurus) armatus, is equally divergent from both GAD65 and GAD67. Specimens of C. (N.) armatus were collected by trawl at a depth of 4,000 m in the Porcupine Seabight (Northeastern Atlantic), and brains dissected and frozen for RNA extraction. All three GAD forms are found in the cerebellum, telencephalon and hypothalamus. Semiquantitative PCR analysis showed that males and females have similar levels of expression of GAD67 and GAD3 in the tissues studied. Independent of the sex examined, the levels of expression of GAD65 and GAD67 in the cerebellum were approximately half that in the telencephalon. GAD3 levels were approximately 30% higher in the cerebellum than in either the telencephalon or hypothalamus. In contrast to GAD67 and GAD3, hypothalamic expression of GAD65 mRNA is 1.8 times higher (p < 0.05) in males than in females. These data indicate that the expression of GAD65, a key enzyme for the synthesis of GABA is sexually dimorphic in females and males of C. (N.) armatus.

  6. Expression of glutamic acid decarboxylase and identification of GABAergic cells in the ischemic rat dentate gyrus

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Dogonowski, Anne-Marie; Finsen, Bente;


    We have investigated the glutamic acid dexcarboxylase (GAD) mRNA and protein isoforms as markers for ischemic loss of GABAergic neurons in the dentate hilus. Stereological counts of these neurons were performed in rats surviving 8 days after 10 min of transient forebrain ischemia, and in control...

  7. Expression of glutamic acid decarboxylase and identification of GABAergic cells in the ischemic rat dentate gyrus

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Dogonowski, Anne-Marie; Finsen, Bente;


    We have investigated the glutamic acid dexarboxylase (GAD) mRNA and protein isoforms as markers for ischemic loss of GABAergic neurons in the dentate hilus. Stereological counts of these neurons were performed in rats surviving 8 days after 10 min of transient forebrain ischemia, and in control...

  8. Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: protean additions to the autoimmune central neuropathies. (United States)

    Ali, Fatima; Rowley, Merrill; Jayakrishnan, Bindu; Teuber, Suzanne; Gershwin, M Eric; Mackay, Ian R


    Stiff Person Syndrome (SPS) is a rare autoimmune neurological disease attributable to autoantibodies to glutamic acid decarboxylase (anti-GAD) more usually associated with the islet beta cell destruction of autoimmune type 1 diabetes (T1D). SPS is characterized by interference in neurons with the synthesis/activity of the inhibitory neurotransmitter gamma amino butyric acid (GABA) resulting in the prototypic progressive spasmodic muscular rigidity of SPS, or diverse neurological syndromes, cerebellar ataxia, intractable epilepsy, myoclonus and several others. Remarkably, a single autoantibody, anti-GAD, can be common to widely different disease expressions, i.e. T1D and SPS. One explanation for these data is the differences in epitope engagement between the anti-GAD reactivity in SPS and T1D: in both diseases, anti-GAD antibody reactivity is predominantly to a conformational epitope region in the PLP- and C-terminal domains of the 65 kDa isoform but, additionally in SPS, there is reactivity to conformational epitope(s) on GAD67, and short linear epitopes in the C-terminal region and at the N-terminus of GAD65. Another explanation for disease expressions in SPS includes ready access of anti-GAD to antigen sites due to immune responsiveness within the CNS itself according to intrathecal anti-GAD-specific B cells and autoantibody. Closer study of the mysterious stiff-person syndrome should enhance the understanding of this disease itself, and autoimmunity in general.


    Directory of Open Access Journals (Sweden)

    N. V. Piven


    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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Production of dopamine by aromatic L-amino acid decarboxylase cells after spinal cord injury

    DEFF Research Database (Denmark)

    Ren, Liqun; Wienecke, Jacob; Hultborn, Hans;


    Aromatic L-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin from 5-hydroxytryptophan after spinal cord injury (SCI)...

  12. Clinical and biochemical features of aromatic L-amino acid decarboxylase deficiency.

    NARCIS (Netherlands)

    Brun, L.; Ngu, L.H.; Keng, W.T.; Ch'ng, G.S.; Choy, Y.S.; Hwu, W.L.; Lee, W.T.; Willemsen, M.A.A.P.; Verbeek, M.M.; Wassenberg, T.; Regal, L.; Orcesi, S.; Tonduti, D.; Accorsi, P.; Testard, H.; Abdenur, J.E.; Tay, S.; Allen, G.F.; Heales, S.; Kern, I.; Kato, M.; Burlina, A.; Manegold, C.; Hoffmann, G.F.; Blau, N.


    OBJECTIVE: To describe the current treatment; clinical, biochemical, and molecular findings; and clinical follow-up of patients with aromatic l-amino acid decarboxylase (AADC) deficiency. METHOD: Clinical and biochemical data of 78 patients with AADC deficiency were tabulated in a database of pediat

  13. Miller-Fisher Syndrome: Are Anti-GAD Antibodies Implicated in Its Pathophysiology?

    Directory of Open Access Journals (Sweden)

    Ioannis E. Dagklis


    Full Text Available Miller-Fisher syndrome (MFS is considered as a variant of the Guillain-Barre syndrome (GBS and its characteristic clinical features are ophthalmoplegia, ataxia, and areflexia. Typically, it is associated with anti-GQ1b antibodies; however, a significant percentage (>10% of these patients are seronegative. Here, we report a 67-year-old female patient who presented with the typical clinical features of MFS. Workup revealed antibodies against glutamic acid decarboxylase (GAD in relatively high titers while GQ1b antibodies were negative. Neurological improvement was observed after intravenous gamma globulin and follow-up examinations showed a continuous clinical amelioration with simultaneous decline of anti-GAD levels which finally returned to normal values. This case indicates that anti-GAD antibodies may be associated with a broader clinical spectrum and future studies in GQ1b-seronegative patients could determine ultimately their clinical and pathogenetic significance in this syndrome.

  14. Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution☆

    Institute of Scientific and Technical Information of China (English)

    Ling Lin; Sheng Hu; Kai Yu; Jun Huang; Shanjing Yao; Yinlin Lei; Guixiang Hu; Lehe Mei


    Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacil us brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L−1·min−1·(mg protein)−1, 120%higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.

  15. Differential expression of glutamic acid decarboxylase in rat and human islets

    DEFF Research Database (Denmark)

    Petersen, J S; Russel, S; Marshall, M O;


    The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat isl...

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


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


    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid seq...

  17. Tissue and regional distribution of cysteic acid decarboxylase. A new assay method. (United States)

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


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

  18. 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: [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)


    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.

  19. 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


    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.

  20. In vivo knockdown of GAD67 in the amygdala disrupts fear extinction and the anxiolytic-like effect of diazepam in mice. (United States)

    Heldt, S A; Mou, L; Ressler, K J


    In mammals, γ-aminobutyric acid (GABA) transmission in the amygdala is particularly important for controlling levels of fear and anxiety. Most GABA synthesis in the brain is catalyzed in inhibitory neurons from L-glutamic acid by the enzyme glutamic acid decarboxylase 67 (GAD67). In the current study, we sought to examine the acquisition and extinction of conditioned fear in mice with knocked down expression of the GABA synthesizing enzyme GAD67 in the amygdala using a lentiviral-based (LV) RNA interference strategy to locally induce loss-of-function. In vitro experiments revealed that our LV-siRNA-GAD67 construct diminished the expression of GAD67 as determined with western blot and fluorescent immunocytochemical analyses. In vivo experiments, in which male C57BL/6J mice received bilateral amygdala microinjections, revealed that LV-siRNA-GAD67 injections produce significant inhibition of endogenous GAD67 when compared with control injections. In contrast, no significant changes in GAD65 expression were detected in the amygdala, validating the specificity of LV knockdown. Behavioral experiments showed that LV knockdown of GAD67 results in a deficit in the extinction, but not the acquisition or retention, of fear as measured by conditioned freezing. GAD67 knockdown did not affect baseline locomotion or basal measures of anxiety as measured in open field apparatus. However, diminished GAD67 in the amygdala blunted the anxiolytic-like effect of diazepam (1.5 mg kg(-1)) as measured in the elevated plus maze. Together, these studies suggest that of GABAergic transmission in amygdala mediates the inhibition of conditioned fear and the anxiolytic-like effect of diazepam in adult mice.

  1. GAD Antibody-Associated Late-Onset Cerebellar Ataxia in Two Female Siblings

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    Joseph Kuchling


    Full Text Available Background: Anti-glutamic acid decarboxylase antibody (GAD-ab-associated cerebellar ataxia is a rare neurological disorder characterized by cerebellar symptoms concomitant with high GAD-ab levels in serum and cerebrospinal fluid (CSF. Case Report: We report on 2 female siblings (aged 74 and 76 years presenting with gradual progression of rotational vertigo, gait ataxia and vertical diplopia, continuously progressing for 6 months and 6 years, respectively. Autoimmune laboratory examinations showed remarkably increased serum and CSF GAD-ab levels. Their medical histories revealed late-onset type 1 diabetes mellitus (T1DM and other concomitant autoimmune disorders (Grave's disease, Hashimoto's thyroiditis. Cerebral MRI and laboratory examinations were unremarkable. The diagnosis of GAD-ab-associated cerebellar ataxia with particular brainstem involvement was established in both women. After the exclusion of an underlying malignancy, immunosuppressive therapy has been initiated in both patients, which resulted in stabilization in one and in clinical improvement in the other patient. Discussion: The unique association of autoantibody-mediated cerebellar ataxia and late-onset T1DM in 2 siblings with similar clinical and paraclinical phenotypes strengthens the concept that hereditary factors might play a relevant role also in autoimmune diseases so far considered to be sporadic. Moreover, the occurrence of continuous vertical diplopia broadens the clinical spectrum of GAD-ab-associated neurological syndromes.

  2. Frequency of islet cell autoantibodies (IA-2 and GAD in young Brazilian type 1 diabetes patients

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    V.C. Pardini


    Full Text Available Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA, anti-insulin, anti-glutamic acid decarboxylase (GAD and the antibody (Ab against tyrosine phosphatase (PTP-like protein known as ICA-512 (IA-2. In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM type 1 patients with recent-onset disease (£12 months and 37 type 1 diabetes patients with long-duration diabetes (>12 months who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0% for GADAb, 62.9% for IA-2Ab and 82.9% for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1% for GADAb and IA-2Ab, and 67.5% for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population.

  3. Selective loss of parvalbumin-positive GABAergic interneurons in the cerebral cortex of maternally stressed Gad1-heterozygous mouse offspring. (United States)

    Uchida, T; Furukawa, T; Iwata, S; Yanagawa, Y; Fukuda, A


    Exposure to maternal stress (MS) and mutations in GAD1, which encodes the γ-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67(+/GFP)) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67(+/GFP) mice. By contrast, these findings were not observed in wild-type (GAD67(+/+)) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.

  4. Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways. (United States)

    Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi


    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.

  5. Cellular target recognition of perfluoroalkyl acids: In vitro evaluation of inhibitory effects on lysine decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sufang; Lv, Qiyan; Yang, Yu, E-mail:; Guo, Liang-Hong, E-mail:; Wan, Bin; Zhao, Lixia


    Perfluoroalkyl acids (PFAAs) have been shown to bind with hepatic peroxisome proliferator receptor α, estrogen receptors and human serum albumin and subsequently cause some toxic effects. Lysine decarboxylase (LDC) plays an important role in cell growth and developmental processes. In this study, the inhibitory effect of 16 PFAAs, including 13 perfluorinated carboxylic acids (PFCAs) and 3 perfluorinated sulfonic acids (PFSAs), on lysine decarboxylase (LDC) activity was investigated. The inhibition constants obtained in fluorescence enzyme assays fall in the range of 2.960 μM to 290.8 μM for targeted PFCAs, and 41.22 μM to 67.44 μM for targeted PFSAs. The inhibitory effect of PFCAs increased significantly with carbon chain (7–18 carbons), whereas the short chain PFCAs (less than 7 carbons) did not show any effect. Circular dichroism results showed that PFAA binding induced significant protein secondary structural changes. Molecular docking revealed that the inhibitory effect could be rationalized well by the cleft binding mode as well as the size, substituent group and hydrophobic characteristics of the PFAAs. At non-cytotoxic concentrations, three selected PFAAs inhibited LDC activity in HepG2 cells, and subsequently resulted in the decreased cadaverine level in the exposed cells, suggesting that LDC may be a possible target of PFAAs for their in vivo toxic effects. - Highlights: • Inhibitory effects of PFAAs on lysine decarboxylase activity were evaluated. • Four different methods were employed to investigate the mechanisms. • The long chain PFAAs showed inhibitory effect compare with 4–6 carbon chain. • The long chain PFAAs bound with LDC differently from the short ones. • The results in cells correlate with those obtained from fluorescence assay.

  6. Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum. (United States)

    Willcutts, M D; Morrison-Bogorad, M


    The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.

  7. 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;


    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....... In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65-/- and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABA......(A) receptor activity. An impaired tonic inhibition in GAD65-/- mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA...

  8. Identification and molecular cloning of glutamate decarboxylase gene from Lactobacillus casei

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    Yasaman Tavakoli


    Full Text Available Gamma-amino butyric acid (GABA possesses several physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects. Production of GABA-enriched products by lactic acid bacteria has been a focus of different researches in recent years because of their safety and health-promoting specifities. In this study, glutamate decarboxylase (gad gene of a local strains Lactobacillus casei was identified and cloned. In order to clone the gad gene from this strain, the PCR was carried out using primers designed based on conserved regions. The PCR product was purified and ligated into PGEM-T vector. Comparison of obtained sequences shows that this fragment codes the pyridoxal 5′-phosphate binding region. This strain could possibly be used for the industrial GABA production and also for development of functional fermented foods. Gad gene manipulation can also either decrease or increase the activity of enzyme in bacteria.

  9. Glutamate Decarboxylase 1 Overexpression as a Poor Prognostic Factor in Patients with Nasopharyngeal Carcinoma (United States)

    Lee, Yi-Ying; Chao, Tung-Bo; Sheu, Ming-Jen; Tian, Yu-Feng; Chen, Tzu-Ju; Lee, Sung-Wei; He, Hong-Lin; Chang, I-Wei; Hsing, Chung-Hsi; Lin, Ching-Yih; Li, Chien-Feng


    Background: Glutamate decarboxylase 1 (GAD1) which serves as a rate-limiting enzyme involving in the production of γ-aminobutyric acid (GABA), exists in the GABAergic neurons in the central nervous system (CNS). Little is known about the relevance of GAD1 to nasopharyngeal carcinoma (NPC). Through data mining on a data set derived from a published transcriptome database, this study first identified GAD1 as a differentially upregulated gene in NPC. We aimed to evaluate GAD1 expression and its prognostic effect on patients with early and locoregionally advanced NPC. Methods: We evaluated GAD1 immunohistochemistry and performed an H-score analysis on biopsy specimens from 124 patients with nonmetastasized NPC receiving treatment. GAD1 overexpression was defined as an H score higher than the median value. The findings of such an analysis are correlated with clinicopathological behaviors and survival rates, namely disease-specific survival (DSS), distant-metastasis-free survival (DMeFS), and local recurrence-free survival (LRFS) rates. Results: GAD1 overexpression was significantly associated with an increase in the primary tumor status (p < 0.001) and American Joint Committee on Cancer (AJCC) stages III-IV (p = 0.002) and was a univariate predictor of adverse outcomes of DSS (p = 0.002), DMeFS (p < 0.0001), and LRFS (p = 0.001). In the multivariate comparison, in addition to advanced AJCC stages III-IV, GAD1 overexpression remained an independent prognosticator of short DSS (p = 0.004, hazard ratio = 2.234), DMeFS (p < 0.001, hazard ratio = 4.218), and LRFS (p = 0.013, hazard ratio = 2.441) rates. Conclusions: Our data reveal that GAD1 overexpression was correlated with advanced disease status and may thus be a critical prognostic indicator of poor outcomes in NPC and a potential therapeutic target to facilitate the development of effective treatment modalities. PMID:27698909

  10. Molecular cloning, expression and in situ hybridization of rat brain glutamic acid decarboxylase messenger RNA. (United States)

    Julien, J F; Legay, F; Dumas, S; Tappaz, M; Mallet, J


    A cDNA library was generated in the expression vector lambda GT11 from rat brain poly(A)+ RNAs and screened with a GAD antiserum. Two clones reacted positively. One of them was shown to express a GAD activity which was specifically trapped on anti-GAD immunogel and was inhibited by gamma-acetylenic-GABA. Blot hybridization analysis of RNAs from rat brain revealed a single 4 kilobases band. Preliminary in situ hybridizations showed numerous cells labelled by the GAD probe such as the Purkinje and stellate cells in the cerebellar cortex and the cells of the reticular thalamic nucleus.

  11. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis. (United States)

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan


    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  12. Cysteamine treatment ameliorates alterations in GAD67 expression and spatial memory in heterozygous reeler mice. (United States)

    Kutiyanawalla, Ammar; Promsote, Wanwisa; Terry, Alvin; Pillai, Anilkumar


    Brain-derived neurotrophic factor (BDNF) signalling through its receptor, TrkB is known to regulate GABAergic function and glutamic acid decarboxylase (GAD) 67 expression in neurons. Alterations in BDNF signalling have been implicated in the pathophysiology of schizophrenia and as a result, they are a potential therapeutic target. Interestingly, heterozygous reeler mice (HRM) have decreased GAD67 expression in the frontal cortex and hippocampus and they exhibit many behavioural and neurochemical abnormalities similar to schizophrenia. In this study, we evaluated the potential of cysteamine, a neuroprotective compound to improve the deficits in GAD67 expression and cognitive function in HRM. We found that cysteamine administration (150 mg/kg.d, through drinking water) for 30 d significantly ameliorated the decreases in GAD67, mature BDNF and full-length TrkB protein levels found in frontal cortex and hippocampus of HRM. A significant attenuation of the increased levels of truncated BDNF in frontal cortex and hippocampus, as well as truncated TrkB in frontal cortex of HRM was also observed following cysteamine treatment. In behavioural studies, HRM were impaired in a Y-maze spatial recognition memory task, but not in a spontaneous alternation task or a sensorimotor, prepulse inhibition (PPI) procedure. Cysteamine improved Y-maze spatial recognition in HRM to the level of wide-type controls and it improved PPI in both wild-type and HRM. Finally, mice deficient in TrkB, showed a reduced response to cysteamine in GAD67 expression suggesting that TrkB signalling plays an important role in GAD67 regulation by cysteamine.

  13. 经皮三叉神经电刺激预处理对戊四氮致痫大鼠海马谷氨酸脱羧酶表达的影响%Effects of pretreatment with transcutaneous trigeminal nerve electrostimulation on expression of glutamic acid decarboxylase in rat hippocampus with pentetrazol-induced seizures

    Institute of Scientific and Technical Information of China (English)

    张慧敏; 李良勇; 李家林; 王玉


    Objective To observe the effects of pretreatment with transcutaneous trigeminal nerve electrostimulation ( TNS ) on expression of glutamic acid decarboxylase( GAD )65/67 in rat hippocampus with pentetrazol-induced seizures and explone its possible anti-epileptic mechanisms. Methods Rats were divided into control group and TNS group which were given pentetrazol( PTZ ) after I ,7.14,28 d consecutive electrostimulation respectively, and subsequently the severity of seizure was quantitatively evaluated within 2 h after intraperitoneal injection of PTZ. The GAD65 and CAD67 protein was analyzed by immunohistochemistry at different time quantitatively in the regions of hippocampus. Results ① Compared with the corresponding control group, the severity of seizure in 14 d and 28 d consecutive TNS groups was significantly milder ( P <0. 05 ), while in I d and 7 d groups, little but no significant change was observed ( P>0. 05 ).② Compared with the corresponding control group, the number of GAD65 posi tive cells in hippocampus was significantly increased in the 7 , 14 ,28 d TNS groups ( P 0.05),TNS组连续刺激14、28 d大鼠癫痫发作程度明显减轻(P0.05),TNS组7、14、28 d大鼠海马区GAD65表达数目明显增加(P<0.05),并且随刺激时间延长,GAD65阳性细胞增加越明显.③ 与相应时间点对照组比较,TNS各组GAD67阳性细胞光密度差异均有统计学意义(P<0.05),但表达增加与刺激时间长短无明显关系.结论 经皮TNS预处理对癫痫发作有一定影响,可能与海马区内GAD65和GAD67阳性表达增多,进而诱导脑内抑制性机制的增强有关.

  14. 谷氨酸脱羧酶抗体微量平板放射结合检测法的建立与初步应用%Micro-plate radiobinding assay of autoantibody to glutamic acid decarboxylase

    Institute of Scientific and Technical Information of China (English)

    黄干; 金河来; 王霞; 李卉; 张松; 周智广


    Objective The purpose of this study was to develop a high-throughput micro-plate radiobinding assay (RBA) of glutamic acid decarboxylase antibody (GAD-Ab) and to evaluate its clinical application. Methods 35labeled GAD65 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℃ PBS buffer, and then counted by a liquid scintillation counter. The GAD-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 TI DM and healthy cases 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 with3 ×104 counts/min 35S-GAD for 24 h under slow vibration, antigen-antibody compounds washed 10 times by 4℃ 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 among 100 healthy

  15. Aromatic Amino Acid Decarboxylase Deficiency Not Responding to Pyridoxine and Bromocriptine Therapy: Case Report and Review of Response to Treatment



    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...

  16. Lack of support for the association between GAD2 polymorphisms and severe human obesity.

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    Michael M Swarbrick


    Full Text Available The demonstration of association between common genetic variants and chronic human diseases such as obesity could have profound implications for the prediction, prevention, and treatment of these conditions. Unequivocal proof of such an association, however, requires independent replication of initial positive findings. Recently, three (-243 A>G, +61450 C>A, and +83897 T>A single nucleotide polymorphisms (SNPs within glutamate decarboxylase 2 (GAD2 were found to be associated with class III obesity (body mass index > 40 kg/m2. The association was observed among 188 families (612 individuals segregating the condition, and a case-control study of 575 cases and 646 lean controls. Functional data supporting a pathophysiological role for one of the SNPs (-243 A>G were also presented. The gene GAD2 encodes the 65-kDa subunit of glutamic acid decarboxylase-GAD65. In the present study, we attempted to replicate this association in larger groups of individuals, and to extend the functional studies of the -243 A>G SNP. Among 2,359 individuals comprising 693 German nuclear families with severe, early-onset obesity, we found no evidence for a relationship between the three GAD2 SNPs and obesity, whether SNPs were studied individually or as haplotypes. In two independent case-control studies (a total of 680 class III obesity cases and 1,186 lean controls, there was no significant relationship between the -243 A>G SNP and obesity (OR = 0.99, 95% CI 0.83-1.18, p = 0.89 in the pooled sample. These negative findings were recapitulated in a meta-analysis, incorporating all published data for the association between the -243G allele and class III obesity, which yielded an OR of 1.11 (95% CI 0.90-1.36, p = 0.28 in a total sample of 1,252 class III obese cases and 1,800 lean controls. Moreover, analysis of common haplotypes encompassing the GAD2 locus revealed no association with severe obesity in families with the condition. We also obtained functional data for the

  17. Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma. (United States)

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


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

  18. Status spasticus and psoas muscle edema due to anti-GAD antibody associated stiff-man syndrome. (United States)

    Maramattom, Boby Varkey


    Severe muscle rigidity and spasms are uncommon causes of Intensive Care Unit (ICU) admissions. Stiff-man syndrome (SMS) is a rare disorder characterized by continuous muscle spasms, axial muscle rigidity, "tin soldier gait," and continuous motor unit activity on electromyography. There are three clinical variants of SMS; stiff-limb syndrome, classical SMS, and paraneoplastic encephalomyelitis with rigidity and myoclonus. Three types of antibodies have been associated with SMS; however, anti-glutamic acid decarboxylase (GAD) antibodies are the most frequent and are seen in the idiopathic type of SMS. The spasms of SMS can be very disabling and severe enough to cause muscle ruptures and skeletal fractures. We present a case of anti-GAD positive SMS with "status spasticus" causing bilateral psoas myoedema and rhabdomyolysis due to repeated axial muscle jerking in a 64-year-old man and discuss the differential diagnosis of a "jerking patient in the ICU."

  19. Glutamic acid decarboxylase 65 autoantibody levels discriminate two subtypes of latent autoimmune diabetes in adults

    Institute of Scientific and Technical Information of China (English)

    李霞; 杨琳; 周智广; 黄干; 颜湘


    Objective To compare the clinical characteristics between type 2 diabetes mellitus (T2DM) and latent autoimmune diabetes in adults (LADA) with different titers of glutamic acid decarboxylase autoantibody (GADA) and to define the two distinct subtypes of LADA.Methods Sera of 750 patients with an initial diagnosis of T2DM from central south of China were screened for GADA using a radioligand assay. The distribution and frequency of GADA levels were described. Two hundred and ninety-five patients were divided into the T2DM group (n=233) and the LADA group (n=62) to compare the age of onset, body mass index, HbA1c, C-peptide, hypertension, dyslipidemia and chronic diabetic complications. Furthermore, LADA patients with different GADA titers were subdivided to analyze the same indexes as the above. Results The prevalence of LADA (defined as GADA≥0.05, namely GADA positive) was 9.7% in the 750 initially diagnosed type 2 diabetic patients. Compared with T2DM, LADA patients were younger at their ages of onset, had lower C-peptide and body mass index, and also had less cases with hypertension and with dyslipidemia. However, only patients with high titer of GADA had poorer beta cell functions and less diabetic complications compared to T2DM and low GADA titer of LADA patients. Patients with low GADA titer were similar to T2DM patients, except that they were prone to develop ketosis more frequently.Conclusions Two clinically distinct subtypes of LADA can be identified by GADA levels in patients initially-diagnosed as type 2 diabetes. Patients with high titer of GADA (GADA≥0.5) subsequently develop more insulin dependency, which are classified as LADA-type 1; while those with lower GADA titer (0.05≤GADA<0.5) and having clinical and metabolic phenotypes of type 2 diabetes are classified as LADA-type 2.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats. (United States)

    Shetty, A K; Turner, D A


    Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the

  2. The application of glutamic acid alpha-decarboxylase for the valorization of glutamic acid

    NARCIS (Netherlands)

    Lammens, T.M.; Biase, De Daniela; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M.


    Glutamic acid is an important constituent of waste streams from biofuels production. It is an interesting starting material for the synthesis of nitrogen containing bulk chemicals, thereby decreasing the dependency on fossil fuels. On the pathway from glutamic acid to a range of molecules, the decar

  3. Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the meninges and the choroid plexus: implications for non-neuronal sources for GABA in the developing mouse brain.

    Directory of Open Access Journals (Sweden)

    Shiro Tochitani

    Full Text Available Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67, both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11-E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.

  4. Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the meninges and the choroid plexus: implications for non-neuronal sources for GABA in the developing mouse brain. (United States)

    Tochitani, Shiro; Kondo, Shigeaki


    Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11-E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.

  5. Cloning, sequence analysis of GAD gene from indica rice and its plant expression vector construction%籼稻GAD基因的克隆、序列分析及其植物表达载体构建

    Institute of Scientific and Technical Information of China (English)

    黄志伟; 许明; 林忠辉; 蔡小玲; 郑金贵


    [目的]克隆籼稻谷氨酸脱羧酶(GAD)基因的全长cDNA,并进行序列分析和植物表达载体构建,为改良水稻的营养保健品质奠定基础.[方法]根据已知植物GAD基因的保守区域设计引物,以高GABA籼稻品系“新-9-4”的胚芽为材料,利用RT-PCR和RACE技术克隆GAD基因的全长cDNA;扩增其编码序列,构建双T-DNA植物表达载体.[结果]扩增获得长1962 bp的籼稻GAD基因全长cDNA(OsiGAD),包含1479 bp的开放阅读框,编码492个氨基酸.OsiGAD与已报道的水稻GAD基因OsGAD1、OsGAD2、OsGAD3的核苷酸序列同源性分别为67.1%、69.4%、98.3%,推导氨基酸序列的同源性分别为77.6%、70.1%、100.0%.OsiGAD蛋白序列具有磷酸吡哆醛结合位点和与钙调蛋白结合的C端延伸区域;构建了其具有水稻胚乳特异表达特性的双T-DNA植物高效表达载体pCDMAR-OsiGAD-hpt,选择标记基因和OsiGAD-因分别位于此载体的两个不同T-DNA区.[结论]克隆获得的籼稻GAD基因OsiGAD长1962 bp,并成功构建了其双T-DNA植物高效表达载体.%[Objective]The current experiment was conducted to clone the full length cDNA of glutamate decarboxy-lase (GAD) gene from indica rice,and then analyze its sequence followed by the construction of plant expression vector to provide references for improving rice nutrition and healthcare quality. [Method]Full length cDNA of GAD gene from indica rice 'Xin-9-4' rich in GABA was cloned by RT-PCR and RACE based on the specific primers of the conserved domain of other GAD genes. Coding sequence was amplified and double T-DNA plant expression vector was constructed. [Result] The amplified full length cDNA (1962 bp) of GAD gene denominated as OsiGAD contained a 1479 bp ORF encoding a 492-amino-acid polypeptide. OsiGAD shared 67.1,69.4 and 98.3% homology with nucleotide sequence of rice OsGADl,OsGAD2 and 0sGAD3,respectively and the deduced amino acid sequence identity was 77.6,70.1 and 100.0%,respectively

  6. C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin. (United States)

    Zik, Moriyah; Fridmann-Sirkis, Yael; Fromm, Hillel


    Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.

  7. Spinal cord hemisection facilitates aromatic L-amino acid decarboxylase cells to produce serotonin in the subchronic but not the chronic phase

    DEFF Research Database (Denmark)

    Azam, Bushra; Wienecke, Jacob; Jensen, Dennis Bo


    Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC) cells to produce...

  8. Garcinol Upregulates GABAA and GAD65 Expression, Modulates BDNF-TrkB Pathway to Reduce Seizures in Pentylenetetrazole (PTZ)-Induced Epilepsy (United States)

    Hao, Fang; Jia, Li-Hua; Li, Xiao-Wan; Zhang, Ying-Rui; Liu, Xue-Wu


    Background Epilepsy is the most predominant neurological disorder characterized by recurrent seizures. Despite treatment with antiepileptic drugs, epilepsy still is a challenge to treat, due to the associated adverse effects of the drugs. Previous investigations have shown critical roles of BDNF-TrkB signalling and expression of glutamic acid decarboxylase 65 (GAD65) and GABAA in the brain during epilepsy. Thus, drugs that could modulate BDNF-TrkB signal and expression of GAD65 and GABAA could aid in therapy. Recent experimental data have focussed on plant-derived compounds in treatments. Garcinol (camboginol), is a polyisoprenylated benzophenone derived from the fruit of Garcinia indica. We investigated the effects of garcinol in pentylenetetrazole (PTZ)-induced epileptic models. Material/Methods Seizure scores were measured in epilepsy kindled mice. Neuronal degeneration and apoptosis were assessed by Nissl staining, TUNEL assay, and Fluoro-Jade B staining. Immunohistochemistry was performed to evaluate cleaved caspase-3 expressions. Expression of BDNF, TrkB, GABAA, GAD65, Bad, Bcl-2, Bcl-xL, and Bax were determined by western blots. Results Significantly reduced seizure scores and mortality rates were observed with pretreatment with garcinol. Elevated expression of apoptotic proteins and caspase-3 in kindled mice were effectively downregulated by garcinol. Epileptogenic mice presented increased BDNF and TrkB with considerably decreased GABAA and GAD65 expression. Garcinol significantly enhanced GABAA and GAD65 while it suppressed BDNF and TrkB. Garcinol enhanced the performance of mice in Morris water maze tests. Conclusions Garcinol exerts neuroprotective effects via supressing apoptosis and modulating BDNF-TrkB signalling and GAD65/GABAA expressions and also enhanced cognition and memory of the mice. PMID:27855137

  9. Neuropsychiatric autoimmune encephalitis without VGKC-complex, NMDAR, and GAD autoantibodies: case report and literature review. (United States)

    Najjar, Souhel; Pearlman, Daniel; Devinsky, Orrin; Najjar, Amanda; Nadkarni, Siddhartha; Butler, Tracy; Zagzag, David


    We report a patient with a seronegative autoimmune panencephalitis, adding a subtype to the emerging spectrum of seronegative autoimmune encephalitis, and we review the sparse literature on isolated psychiatric presentations of autoimmune encephalitis. (A PubMed search for "seronegative autoimmune encephalitis," "nonvasculitic autoimmune inflammatory meningoencephalitis," and related terms revealed VGKC)-complex, N-methyl-D-aspartate receptor (NMDAR), and glutamic acid decarboxylase (GAD) autoantibodies. We excluded genetic, metabolic, paraneoplastic, degenerative, and infectious etiologies. The patient's symptoms remitted fully with immune therapy, but recurred in association with widespread bihemispheric brain lesions. Brain biopsy revealed mild nonvasculitic inflammation and prominent vascular hyalinization. Immune therapy with plasma exchanges cleared the MRI abnormalities but, 10 years after onset, the patient still suffers neuropsychiatric sequelae. We conclude that autoimmune panencephalitis seronegative for VGKC-complex, NMDAR, and GAD autoantibodies is a subtype of autoimmune encephalitis that can present with pure neuropsychiatric features and a normal brain MRI. Immunologic mechanisms may account for psychiatric symptoms in a subset of patients now diagnosed with classical psychotic disorders. Delay in starting immune therapy can lead to permanent neuropsychiatric sequelae. We propose a standardized classification system for the autoimmune encephalitides, integrating earlier pathology-oriented terms with more recently defined serologic and clinical phenotypes.

  10. GAD65基因修饰的神经干细胞移植治疗癫痫的实验研究%Transplantation of GAD65 - engineered neural stem cells for treatment of epilepsy:A study in rat model

    Institute of Scientific and Technical Information of China (English)

    刘霞; 齐志国; 谢鹏


    目的:将谷氨酸脱羧酶65(GAD65)基因修饰的神经干细胞移植到海仁酸颞叶癫痫模型大鼠海马中,检测目的基因在脑内的表达以及基因工程化细胞移植对癫痫的影响.方法:建立海仁酸致癫痫大鼠模型,完全随机分为GAD-NSCs移植组、单纯NSCs移植组、生理盐水对照组,每组20只.在移植后1、2、3天,1、2、4、8周各时间点,通过小剂量戊四氮诱发试验检测大鼠的癫痫敏感性;移植1周后,取海马进行WestemBlotting检测GAD65表达;移植8周后,取脑组织,进行海马区HE病理染色及BrdU和GAD5的免疫组化染色.结果:移植后,GAD-NSCs移植组2天就表现出癫痫敏感性下降,而单纯NSCs移植组术后2周才出现该变化,并且生理盐水对照组和单纯NSCs移植组Racine评分标准评分高于GAD-NSCs称植组;Westem Blotting检测表明仅有CAD-NSCs移植组海马区GAD65高表达;GAD-NSCs移植组的BrdU阳性标记细胞、GAD65阳性标记细胞均显著高于其他两组.结论:GAD65基因修饰神经干细胞移植到癫痫大鼠海马中,海马区GAD5呈高表达,可持续抑制实验动物癫痫敏感性,并对移植后的宿主神经干细胞具有保护作用,改善实验动物神经病理变化.%Objective: To analyze expression of glutamic acid decarboxylase - 65(GADs65) in brain and therapeutic effects of GAD65 - engeneered neural stem cells after the cells being transplanated into hippocampus of kainic acid- induced epilepsy model rats. Methods: Kainic acid- induced epilepsy model rats were divided into 3 groups completely randomly: GAD- NSCs transplantation group (n = 20), NSCs transplantation group (n = 20), saline control group(n = 20).The sensitivity in epilepsy models was detected by small dosed pentetrazole - induced test corresponding to 1, 2 and 3 days, 1, 2, 4 and 8 weeks after transplantation respectively. GAD65 expression in hippocampus was examined by Western Blotting at 1 week post- transplantation. Hematoxylin - eosin

  11. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants. (United States)

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H


    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

  12. Correlations of Clusters of Non-Convulsive Seizure and Magnetic Resonance Imaging in a Case With GAD65-Positive Autoimmune Limbic Encephalitis. (United States)

    Gardner, Rachael; Rangaswamy, Rajesh; Peng, Yen-Yi


    With the increased availability of laboratory tests, glutamic acid decarboxylase (GAD) antibody-positive limbic encephalitis has become an emerging diagnosis. The myriad symptoms of limbic encephalitis make the diagnosis challenging. Symptoms range from seizures, memory loss, dementia, confusion, to psychosis. We present a case of a 21-year-old female with GAD65 antibody-positive limbic encephalitis. The case is unique because the clinical course suggests that non-convulsive seizures are the major cause of this patient's clinical manifestations. The following is the thesis: systemic autoimmune disease, associated with the GAD65 antibody, gives rise to seizures, in particular, non-convulsive seizures. Temporal lobes happen to be the most susceptible sites to develop seizures. The greater part of these seizures can be non-convulsive and hard to recognize without electroencephalogram (EEG) monitoring. The variable symptoms mirror the severity and locations of these seizures. The magnetic resonance imaging (MRI) signal abnormities in the bilateral hippocampus, fornix, and mammillary body correlate with the density of these seizures in the similar manner, which suggests it is secondary to post-ictal edema.

  13. Escherichia coli K-12 survives anaerobic exposure at pH 2 without RpoS, Gad, or hydrogenases, but shows sensitivity to autoclaved broth products.

    Directory of Open Access Journals (Sweden)

    Daniel P Riggins

    Full Text Available Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS, cyclopropane fatty acid synthase (Cfa and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2-pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating in a glove box excluding oxygen (10% H2, 5% CO2, balance N2. With dissolved O2 concentrations maintained below 6 µM, survival at pH 2 required Cfa but did not require GadC, RpoS, or hydrogenases. Extreme-acid survival in broth (containing tryptone and yeast extract was diminished in media that had been autoclaved compared to media that had been filtered. The effect of autoclaved media on extreme-acid survival was most pronounced when oxygen was excluded. Exposure to H2O2 during extreme-acid treatment increased the death rate slightly for W3110 and to a greater extent for the rpoS deletion strain. Survival at pH 2 was increased in strains lacking the anaerobic regulator fnr. During anaerobic growth at pH 5.5, strains deleted for fnr showed enhanced transcription of acid-survival genes gadB, cfa, and hdeA, as well as catalase (katE. We show that E. coli cultured under oxygen exclusion (<6 µM O2 requires mechanisms different from those of aerated cultures. Extreme acid survival is more sensitive to autoclave products under oxygen exclusion.

  14. Differential Regulation of Glutamic Acid Decarboxylase Gene Expression after Extinction of a Recent Memory vs. Intermediate Memory (United States)

    Sangha, Susan; Ilenseer, Jasmin; Sosulina, Ludmila; Lesting, Jorg; Pape, Hans-Christian


    Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65…

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

    NARCIS (Netherlands)

    Romagnoli, G.; Luttik, M.A.H.; Kötter, P.; Pronk, J.T.; Daran, J.M.


    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share seque

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

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


    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.

  17. Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction. (United States)

    Yip, Jane; Soghomonian, Jean-Jacques; Blatt, Gene J


    It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

  18. Early development of GABAergic cells of the retina in sharks: an immunohistochemical study with GABA and GAD antibodies. (United States)

    Ferreiro-Galve, Susana; Candal, Eva; Carrera, Iván; Anadón, Ramón; Rodríguez-Moldes, Isabel


    We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27-29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.

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

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


    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.

  20. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomycescerevisiae

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    Kamei, Yuka; Tamura, Takayuki [Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Yoshida, Ryo [Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ohta, Shinji [Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Fukusaki, Eiichiro [Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Mukai, Yukio, E-mail: [Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)


    Highlights: {yields}We demonstrate that two genes in the yeast GABA metabolism pathway affect aging. {yields} Deletion of the UGA1 or GAD1 genes extends replicative lifespan. {yields} Addition of GABA to wild-type cultures has no effect on lifespan. {yields} Intracellular GABA levels do not differ in longevity mutants and wild-type cells. {yields} Levels of tricarboxylic acid cycle intermediates positively correlate with lifespan. -- Abstract: Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for {gamma}-aminobutyric acid (GABA)-dependent induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The {Delta}uga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for {Delta}uga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of {sup 1}H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with lifespan

  1. Number and regional distribution of GAD65 mRNA-expressing interneurons in the rat hippocampal formation. (United States)

    Czéh, B; Abrahám, Hajnalka; Tahtakran, Siroun; Houser, Carolyn R; Seress, L


    In rodent models for neuropsychiatric disorders reduced number of hippocampal interneurons have been reported, but the total number of GABAergic neurons in the normal rat hippocampus is yet unknown. We used in situ hybridization method to label the 65 isoform of glutamic acid decarboxylase (GAD65) and counted the number of GAD65 mRNA-expressing neurons along the entire septo-temporal axis of the hippocampus. We found that 2/3 of the interneurons were in Ammon's horn (61,590) and 1/3 in the dentate gyrus (28,000). We observed the following numbers in Ammon's horn: CA3 area 33,400, CA2 area 4,190, CA1 area 24,000 and in the dentate gyrus: 6,000 in the molecular and 9,000 in the granule cell layers and 13,000 in the hilus. GAD65 mRNA-expressing neurons were significantly more numerous in dorsal than in ventral hippocampus. The ratio between interneurons and principal cells was lowest in the granule cell layer (0.9%) and highest in hilus (21%). In Ammon's horn this ratio was constant being 13% in CA3 and 8% in CA1-2 areas. In the entire hippocampal formation, the interneuron/principal cell ratio was 6%, with a significant difference between Ammon's horn (9.5%) and the dentate gyrus (2.8%) including the hilus. Such low ratios could suggest that even a limited loss of GABAergic neurons in the hippocampus may have a considerable functional impact.

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

    Roy, Ajit; Ranjan, Akash


    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.

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

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    Alghadir AH


    Full Text Available Ahmad H Alghadir,1 Sami A Gabr,1,2 Einas Al-Eisa11Department of Rehabilitation Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia; 2Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, EgyptBackground: Homeostatic imbalance of trace elements such as iron (Fe, copper (Cu, and zinc (Zn demonstrated adverse effects on brain function among older adults.Objective: The present study aimed to investigate the effects of trace elements and the presence of anti-glutamic acid decarboxylase antibodies (GADAs in human cognitive abilities among healthy older adults.Methods: A total of 100 healthy subjects (65 males, 35 females; age range; 64–96 years were recruited for this study. Based on Loewenstein Occupational Therapy Cognitive Assessment (LOTCA score, the participants were classified according to cognitive performance into normal (n=45, moderate (n=30, and severe (n=25. Cognitive functioning, leisure-time physical activity (LTPA, serum trace elements – Fe, Cu, Zn, Zn/Cu, and GADAs were assessed using LOTCA battery, pre-validated physical activity (PA questionnaire, atomic absorption, and immunoassay techniques, respectively.Results: Approximately 45% of the study population (n=45 had normal distribution of cognitive function and 55% of the study population (n=55 had abnormal cognitive function; they were classified into moderate (score 62–92 and severe (score 31–62. There was a significant reduction in the level of Zn and Zn/Cu ratio along with an increase in the level of Fe, Cu, and anti-GADAs in subjects of severe (P=0.01 and moderate (P=0.01 cognitive performance. LOTCA-cognitive scores correlated positively with sex, HbA1c, Fe, Cu, Zn, and Zn/Cu ratio, and negatively with age, PA, body mass index, and anti-GADAs. Significant inter-correlation was reported between serum trace element concentrations and anti-GADAs which suggest producing a cognitive decline via oxidative and neural

  4. Clinical and genetic characteristics of anti-gad positive type 2 diabetes in Japanese

    Institute of Scientific and Technical Information of China (English)


    Antibodies to glutamic acid decarboxylase (GADab) is considered to be a marker for the autoimmune process against pancreatic β cells. Indeed, nearly 70% of patients with type 1 diabetes is repoted to be GAD ab+. A subgroup of patients diagnosed as type 2 diabetes has GADab. Therefore, it is questioned whether GADab+ patients with type 2 diabetes represent a late onset of type 1 diabetes or a unique disease entity. Fifty five GADab+ patients with type 2 diabetes were compared with 137 GADab- patients. They were admitted to Abe Diabetes Clinic for the control of diabetes. The age at onset of these patients was >30 years, and did not require insulin therapy for at least 6 months from the disease onset. The GADab+ patients had lower urinary C-peptide concentrations (uCPR) [(47.8±48.9) μg/d vs (58.1±49.9) μg/d, P=0.034] than the GADab- patients. The GAD+ patients were assigned insulin therapy more often (81.8% vs 56.3%, P=0.0038) and earlier [(8.5±7.5)years vs (10.1±7.3) years, P=3.3×10-12] than the GAD- patients. The levels of uCPR were associated with the titers of GADab (r=0.32, P=0.038). Among the susceptible HLA alleles for type 1 diabetes, the frequencies of B54 and DRB1* 0405 alleles, but not B61 and DRB1*0901 alleles, were increased among GADab+ patients. There data suggest that the GADab+ type 2 diabetes has an autoimmune nature, although the extent of insulin dependency and the distribution of HLA susceptible alleles are different from type 1 diabetes.

  5. Sex-specific impairment and recovery of spatial learning following the end of chronic unpredictable restraint stress: potential relevance of limbic GAD. (United States)

    Ortiz, J Bryce; Taylor, Sara B; Hoffman, Ann N; Campbell, Alyssa N; Lucas, Louis R; Conrad, Cheryl D


    Chronic restraint stress alters hippocampal-dependent spatial learning and memory in a sex-dependent manner, impairing spatial performance in male rats and leaving intact or facilitating performance in female rats. Moreover, these stress-induced spatial memory deficits improve following post-stress recovery in males. The current study examined whether restraint administered in an unpredictable manner would eliminate these sex differences and impact a post-stress period on spatial ability and limbic glutamic acid decarboxylase (GAD65) expression. Male (n=30) and female (n=30) adult Sprague-Dawley rats were assigned to non-stressed control (Con), chronic stress (Str-Imm), or chronic stress given a post-stress recovery period (Str-Rec). Stressed rats were unpredictably restrained for 21 days using daily non-repeated combinations of physical context, duration, and time of day. Then, all rats were tested on the radial arm water maze (RAWM) for 2 days and given one retention trial on the third day, with brains removed 30min later to assess GAD65 mRNA. In Str-Imm males, deficits occurred on day 1 of RAWM acquisition, an impairment that was not evident in the Str-Rec group. In contrast, females did not show significant outcomes following chronic stress or post-stress recovery. In males, amygdalar GAD65 expression negatively correlated with RAWM performance on day 1. In females, hippocampal CA1 GAD65 positively correlated with RAWM performance on day 1. These results demonstrate that GABAergic function may contribute to the sex differences observed following chronic stress. Furthermore, unpredictable restraint and a recovery period failed to eliminate the sex differences on spatial learning and memory.

  6. Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice. (United States)

    Shimizu, Tokiko; Hayashi, Yoshinori; Yamasaki, Ryo; Yamada, Jun; Zhang, Jian; Ukai, Kiyoharu; Koike, Masato; Mine, Kazunori; von Figura, Kurt; Peters, Christoph; Saftig, Paul; Fukuda, Takaichi; Uchiyama, Yasuo; Nakanishi, Hiroshi


    Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms

  7. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot]. (United States)

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe


    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  8. Anti-Yo and anti-glutamic acid decarboxylase antibodies presenting in carcinoma of the uterus with paraneoplastic cerebellar degeneration: a case report

    Directory of Open Access Journals (Sweden)

    Panegyres Peter K


    Full Text Available Abstract Introduction Paraneoplastic cerebellar degeneration is a rare non-metastatic manifestation of malignancy. In this report, to the best of our knowledge we describe for the first time a diagnosis of paraneoplastic cerebellar degeneration several months prior to the diagnosis of clear carcinoma of the uterus. Case presentation A 75-year-old Caucasian woman manifested a rapidly progressive cerebellar syndrome with nystagmus, past-pointing, dysdiadochokinesis, dysarthria, truncal ataxia and titubation. The paraneoplastic cerebellar degeneration was associated with anti-Yo and anti-glutamic acid decarboxylase antibodies. 14-3-3 protein was detected in the cerebrospinal fluid. She was treated with intravenous immunoglobulin prior to laparotomy, hysterectomy and bilateral salpingoophorectomy. Our patient has survived for three years following diagnosis and treatment. Conclusions To the best of our knowledge this is the first report of an association of clear cell carcinoma of the uterus and paraneoplastic cerebellar degeneration with both anti-Yo and anti-glutamic acid decarboxylase antibodies. The findings imply that both antibodies contributed to the fulminating paraneoplastic cerebellar degeneration observed in our patient, and this was of such severity it resulted in the release of 14-3-3 protein in the cerebrospinal fluid, a marker of neuronal death.

  9. 4-Vinylphenol biosynthesis from cellulose as the sole carbon source using phenolic acid decarboxylase- and tyrosine ammonia lyase-expressing Streptomyces lividans. (United States)

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


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

  10. Impact of Cell-free Supernatant of Lactic Acid Bacteria on Putrescine and Other Polyamine Formation by Foodborne Pathogens in Ornithine Decarboxylase Broth. (United States)

    Ozogul, Fatih; Tabanelli, Giulia; Toy, Nurten; Gardini, Fausto


    Conversion of ornithine to putrescine by Salmonella Paratyphi A, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli was investigated in ornithine decarboxylase broth (ODB) using cell-free supernatants (CFSs) obtained from Leuconostoc mesenterodies subsp. cremoris, Pediococcus acidilactici, Lactococcus lactis subsp. lactis, Streptococcus thermophilus. Two groups of cell-free supernatants (25 or 50%) and control (only ODB) were prepared to investigate putrescine (PUT) and other polyamine formation by foodborne pathogens (FBPs). Significant differences (p < 0.05) were observed among the species for each amine. All of the CFSs reduced the formation of PUT by ≥65%. The production of cadaverine (CAD) was scarcely affected by the presence of CFSs, with the exception of the samples inoculated with L. monocytogenes. The variation in polyamine was found with respect to the control samples. Spermidine (SPD) was produced in lower amount in many samples, especially in Gram-negative FBPs, whereas spermine (SPN) increased drastically in the major part of the samples concerning the control. Histamine (HIS) was characterized by a marked concentration decrease in all of the samples, and tyramine (TYR) was accumulated in very low concentrations in the controls. Therefore, the ability of bacteria to produce certain biogenic amines such as HIS, TYR, PUT, and CAD has been studied to assess their risk and prevent their formation in food products. The results obtained from this study concluded that the lactic acid bacteria (LAB) strains with non-decarboxylase activity are capable of avoiding or limiting biogenic amine formation by FBP.

  11. 水杨酸钠对幼年和成年豚鼠听性脑干反应阈值及螺旋神经节谷氨酸脱羧酶表达的影响%Effect of sodium salicylate on the auditory brain stem response threshold and expression of glutamic acid decarboxylase in spiral ganglion of juvenile and adult guinea pigs

    Institute of Scientific and Technical Information of China (English)

    尹时华; 唐安洲; 谭颂华; 陈平; 谢利红; 任毅


    目的 观察水杨酸钠对幼年和成年豚鼠听性脑干反应(ABR)阈值以及螺旋神经节谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)表达的影响.方法 选择出生4 d的豚鼠40只和出生后1个月的成年豚鼠40只,分成4组(每组20只):幼年对照组,成年对照组,幼年水杨酸钠给药组[300 mg/(kg·d)],成年水杨酸钠给药组[300 mg/(kg·d)].给药15 d后每组随机选取10只豚鼠检测ABR阈值,采用免疫组织化学染色方法检测螺旋神经节GAD的表达.各组剩下的动物停止给药,继续喂养30 d后检测ABR阈值和螺旋神经节GAD的表达.结果 给药15 d后幼年水杨酸钠给药组和成年水杨酸钠给药组豚鼠ABR阈值较给药前以及同期对照组均有提高(P值均<0.001),停药30 d后幼年水杨酸钠给药组ABR阈值恢复到给药前水平,而成年水杨酸钠给药组仍停留在高阈值水平;水杨酸钠给药15 d后能明显下调幼年以及成年豚鼠螺旋神经节GAD蛋白表达,幼年豚鼠GAD表达水平低于成年豚鼠(t=4.7,P<0.001),停药30 d后幼年给药组螺旋神经节GAD表达恢复到同期对照组水平,而成年给药组则继续停留在低表达水平.结论 水杨酸钠在对幼年和成年豚鼠ABR阚值以及螺旋神经节GAD表达的影响上存在差异,其对幼年豚鼠的影响更为明显,但停药后幼年豚鼠较成年豚鼠更容易恢复到正常水平.%Objective To study the differences of regulation of sodium salicylate on the auditory brain stem response(ABR)threshold and expression of glutamic acid decarboxylase(GAD)protein in spiral ganglion of juvenile and adult guinea pigs.Methods Fourty juvenile guinea pigs which were born just four days and fourty adult guinea pigs which were born thirty days were selected.They were divided four groups (group A;group B;group C;group D).ABR threshold was detected before administration.after administration for 15 days and after administration stopped for 30 days.The protein expression of GAD

  12. 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


    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.

  13. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter. (United States)

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


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

  14. Spinal cord hemisection facilitates aromatic L-amino acid decarboxylase cells to produce serotonin in the subchronic but not the chronic phase

    DEFF Research Database (Denmark)

    Azam, Bushra; Wienecke, Jacob; Jensen, Dennis Bo;


    12) were used with a postoperation interval at 5 days or 60 days. Using immunohistochemistry, first, we observed a significant reduction in the density of 5-HT-immunoreactive fibers in the spinal cord below the lesion on the injured side for both groups. Second, we found that the AADC cells were......Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC) cells to produce...... 5-HT from its precursor (5-hydroxytryptophan, 5-HTP) is dramatically increased following complete spinal cord transection. In this study, we investigated whether a partial loss of 5-HT innervation could similarly increase AADC activity. Adult rats with spinal cord hemisected at thoracic level (T11/T...

  15. Synergistic and antagonistic effect of lactic acid bacteria on tyramine production by food-borne pathogenic bacteria in tyrosine decarboxylase broth. (United States)

    Kuley, Esmeray; Ozogul, Fatih


    The effect of lactic acid bacteria (LAB) strains on tyramine (TYR) and also other biogenic amines (BA) production by eight common food-borne pathogen (FBP) in tyrosine decarboxylase broth (TDB) was investigated by using a rapid HPLC method. Significant differences were observed among the FBP strains in ammonia (AMN) and BA production apart from tryptamine, histamine (HIS) and spermine formation (pfood-borne pathogenic bacteria, although the effect of some LAB strains on BA production was strain-dependent. Lactococcus spp. and Streptococcus spp. resulted in significantly higher TYR accumulation by Aeromonas hydrophila and Enterococcus faecalis in TDB. The presence of Lactococcus and/or Lactobacillus in TDB significantly increased HIS production by A. hydrophila, Escherichia coli, Ent. faecalis, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas HIS accumulation was significantly reduced by Staphylococcus aureus, S. paratyphi A and Listeria monocytogenes.

  16. 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

    Directory of Open Access Journals (Sweden)

    Juri-Alexander eWitt


    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

  17. Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

    Directory of Open Access Journals (Sweden)

    Osamu Kakinohana

    Full Text Available BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B receptor agonist, while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD rats were exposed to transient spinal ischemia (10 min to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can

  18. Anti glutamate-decarboxylase antibodies: a liaison between localisation related epilepsy, stiff-person syndrome and type-1 diabetes mellitus. (United States)

    Szűcs, Anna; Barcs, Gábor; Winkler, Gábor; Soós, Zsuzsanna; Folyovich, András; Kelemen, Anna; Várallyay, Péter; Kamondi, Anita


    We present two patients with partial epilepsy, type-1 diabetes and stiff person syndrome associated with high serum auto-antibody levels to glutamate-decarboxylase (anti-GAD). Both patients were or have suffered from additional autoimmune conditions. The presence of stiff person syndrome and elevated anti-GAD levels have to make clinicians look for additional autoimmune conditions including type-1 diabetes. On the other hand, the co-morbidity of partial epilepsy with autoimmune conditions in patients with elevated serum anti-GAD suggests an autoimmune mechanism of partial epilepsy in these cases.

  19. Changes in GAD67 mRNA expression evidenced by in situ hybridization in the brain of R6/2 transgenic mice. (United States)

    Gourfinkel-An, I; Parain, K; Hartmann, A; Mangiarini, L; Brice, A; Bates, G; Hirsch, E C


    Huntington's disease is an autosomal dominant disorder with degeneration of medium size striatal neurones. As the disease evolves, other neuronal populations are also progressively affected. A transgenic mouse model of the disease (R6/2) that expresses exon 1 of the human Huntington gene with approximately 150 CAG repeats has been developed, but GABA concentrations are reported to be normal in the striatum of these animals. In the present study, we analysed the status of GABAergic systems by means of glutamic acid decarboxylase (GAD)67 mRNA in situ hybridization in the brain of R6/2 transgenic mice and wild-type littermates. We show that GAD67 expression is normal in the striatum, cerebellum and septum but decreased in the frontal cortex, parietal cortex, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata of R6/2 mice. These data, which may, in part, account for the behavioural changes seen in these animals, indicate that at 12.5 weeks of age the pathological features seen in the mice differ from those seen in humans with Huntington's disease.

  20. Application of Glutamic Acid Decarboxylase Antibody Test in Children with Diabetes%谷氨酸脱羧酶抗体检测在儿童糖尿病中的应用

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the application value of detection of glutamic acid decarboxylase antibody in children with diabetes in. Methods Select the January 2013, 2015 years 1 month in the hospital of glutamic acid decarboxylase antibody detection of 97 cases of diabetes mellitus patients as the observation group, and the selection of 97 cases of normal children as control group, on the two groups of glutamic acid decarboxylase antibody positive rate. Results After testing, observation group of glutamic acid decarboxylase antibody positive rate was 97.9%, control group of glutamic acid decarboxylase anti-body positive rate was 0, two groups compared with statistical significance (P<0.05). Conclusion Glutamic acid decarboxy-lase antibody detection of children with type 1 diabetes diagnosis has important clinical significance, it is worth in the clini-cal application.%目的:探讨谷氨酸脱羧酶抗体检测在儿童糖尿病中的应用价值。方法选取2013年1月-2015年1月在医院进行谷氨酸脱羧酶抗体检测的97例糖尿病患儿作为观察组,并选取97例正常儿童作为对照组,对两组谷氨酸脱羧酶抗体阳性率做比较。结果经过检测后,观察组谷氨酸脱羧酶抗体阳性率为97.9%,对照组谷氨酸脱羧酶抗体阳性率为0,两组比较差异具有统计学意义(P<0.05)。结论进行谷氨酸脱羧酶抗体检测对儿童1型糖尿病的诊断具有重大的临床意义,值得在临床上推广应用。


    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  2. Styrene production from a biomass-derived carbon source using a coculture system of phenylalanine ammonia lyase and phenylacrylic acid decarboxylase-expressing Streptomyces lividans transformants. (United States)

    Fujiwara, Ryosuke; Noda, Shuhei; Tanaka, Tsutomu; Kondo, Akihiko


    To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S. lividans, and the resulting S. lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S. lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S. lividans/FDC1 was cultured together with S. lividans/p-encP, which we previously reported as a CA-producing S. lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively.

  3. 一种谷氨酸脱羧酶65相关肽融合蛋白的制备及其治疗1型糖尿病的药效研究%Genetic Fusion of Glutamic Acid Decarboxylase 65 Derived Peptides to the B-Subunit of Cholera Toxin and Its Retardation Effect of Diabetes in NOD Mice

    Institute of Scientific and Technical Information of China (English)

    王华倩; 张会勇; 杨洁; 鲁勇; 李泰明; 金亮; 曹荣月; 刘景晶


    使用基因工程方法构建了霍乱毒素B亚单位(Cholera toxin B subunit,CTB)与谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)串联三肽GADⅢ(包括p217-236,p524-538,p290-306)合基因CTB-GADⅢ.将融合基因克隆到大肠杆菌表达载体pET-28a中,获得的重组质粒转化大肠杆菌BL21(DE3).重组菌株经乳糖诱导后,其表达产物经过15%SDS-PAGE分析表明该菌株可以以包涵体形式表达融合蛋白,Mr约为17.6 k.含有CTB-GADⅢ重组蛋白的包涵体经过变性、复性、纯化后,可以得到五聚体结构的CTB-GADⅢ.神经节苷脂GM1(monosialoganglioside)结合实验表明重组CTB-GADⅢ蛋白可以与GM1特异性结合,表明该融合蛋白保持了CTB形成五聚体的生物活性.使用该重组蛋白在NOD小鼠周龄、10周龄和12周龄时滴鼻免疫小鼠共3次,可以显著降低小鼠的发病率,达到治疗1型糖尿病的作用.

  4. Expression of messenger RNAs for glutamic acid decarboxylase, preprotachykinin, cholecystokinin, somatostatin, proenkephalin and neuropeptide Y in the adult rat superior colliculus. (United States)

    Harvey, A R; Heavens, R P; Yellachich, L A; Sirinathsinghji, D J


    The mammalian superior colliculus is an important subcortical integrator of sensorimotor behaviours. It is multi-layered, each layer containing specific neuronal types and possessing distinct input/output relationships. Here we use in situ hybridisation methods to map the distribution of seven neurotransmitters/neuromodulator systems in adult rat superior colliculus. Coronal sections were probed for preprotachykinin, cholecystokinin, somatostatin, proenkephalin, neuropeptide Y and the enzymes glutamic acid decarboxylase and choline acetyltransferase, markers for GABA and acetylcholine respectively. Cells expressing glutamic acid decarboxylase messenger RNA were the most abundant, the highest density being found in the superficial layers. Many cells containing proprotachykinin messenger RNA were found in stratum zonale and the upper two-thirds of stratum griseum superficiale; cells were also located in deeper tectal laminae, particularly caudomedially. Most cholecystokinin messenger RNA expressing cells were located in the superficial layers with a prominent band in the middle third of stratum griseum superficiale. Cells expressing moderate to high levels of somatostatin messenger RNA formed a dense band in the lower third of stratum griseum superficiale/upper stratum opticum; two less distinct tiers of labelling were seen in deeper layers. These in situ hybridisation data reveal three distinct sub-laminae in rat stratum griseum superficiale. Cells expressing moderate to low levels of proenkephalin messenger RNA were located in lower stratum griseum superficiale/upper stratum opticum and intermediate laminae. A cluster of enkephalinergic cells was located medially in the deep tectal laminae. Expression of neuropeptide Y messenger RNA was relatively low and mostly confined to cells in stratum griseum superficiale and stratum opticum. No choline acetyltransferase messenger RNA was detected. This in situ analysis of seven different neurotransmitters

  5. Spinal Cord Hemisection Facilitates Aromatic L-Amino Acid Decarboxylase Cells to Produce Serotonin in the Subchronic but Not the Chronic Phase

    Directory of Open Access Journals (Sweden)

    Bushra Azam


    Full Text Available Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC cells to produce 5-HT from its precursor (5-hydroxytryptophan, 5-HTP is dramatically increased following complete spinal cord transection. In this study, we investigated whether a partial loss of 5-HT innervation could similarly increase AADC activity. Adult rats with spinal cord hemisected at thoracic level (T11/T12 were used with a postoperation interval at 5 days or 60 days. Using immunohistochemistry, first, we observed a significant reduction in the density of 5-HT-immunoreactive fibers in the spinal cord below the lesion on the injured side for both groups. Second, we found that the AADC cells were similarly expressed on both injured and uninjured sides in both groups. Third, increased production of 5-HT in AADC cells following 5-HTP was seen in 5-day but not in 60-day postinjury group. These results suggest that plastic changes of the 5-HT system might happen primarily in the subchronic phase and for longer period its function could be compensated by plastic changes of other intrinsic and/or supraspinal modulation systems.

  6. Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos. (United States)

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei


    We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

  7. Induction of aromatic L-amino acid decarboxylase mRNA by interleukin-1 beta and prostaglandin E2 in PC12 cells. (United States)

    Li, X M; Juorio, A V; Boulton, A A


    Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1-beta and prostaglandin (PG) E2 in PC12 cells. The cells were treated with different doses of IL 1-beta and PGE2 for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1-beta and PGE2 produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE2 formation, its effects may be occurring by increasing level of PGE2.

  8. High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process. (United States)

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


    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.

  9. The influence of the cell free solution of lactic acid bacteria on tyramine production by food borne-pathogens in tyrosine decarboxylase broth. (United States)

    Toy, Nurten; Özogul, Fatih; Özogul, Yesim


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


    Institute of Scientific and Technical Information of China (English)

    WUGuoqi; LINGDaren; 等


    The preparation and characterization of an immobilized L-glutamic decarboxylase(GDC) were studied.This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor,which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin(carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode.The conditions for the enzyme immobilization were optimized by the parameters:buffer composition and concentration,adsorption equilibration time,amount of enzyme,temperature,ionic strength and pH.The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial ate of the enzyme reaction,the effect of various parameters on the immobilized GDC activity and its stability.An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid.The limit of detection is 1.0×10-5M.The linearity response is in the range of 5×10-2-5×10-5M.The equation of linear regression of the calibration curve is y=43.3x+181.6(y is the milli-volt of electrical potential response,x is the logarithm of the concentration of the substrate of L-glutamate acid).The correlation coefficient equals 0.99.The coefficient of varioation equals 2.7%.

  11. Analysis list: Gad1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Gad1 Pluripotent stem cell + mm9

  12. The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency. (United States)

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


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

  13. 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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  14. 老年糖尿病患者血清GAD-Ab、ICA、IAA和IA-2-Ab联合检测的临床应用价值%Clinical Value of Combined Detection of GAD-Ab, ICA, IAA and IA-2-Ab in Elderly Patients with Diabetes Mellitus

    Institute of Scientific and Technical Information of China (English)

    周细国; 杨一波; 杨伟平


    [目的]探讨谷氨酸脱羧酶抗体(GAD-Ab)、胰岛细胞抗体(ICA)、胰岛自身抗体(IAA)和蛋白酪氨酸磷酸酶抗体(IA-2-Ab)联合检测对老年糖尿病患者的应用价值.[方法]采用免疫印迹法检测330例老年糖尿病患者和40例正常人血清中GAD-Ab、ICA、IAA和IA-2-Ab,并对四项自身抗体全阴患者与有一项以上阳性患者的空腹血糖(FBG)、餐后两小时血糖(2hPBG)和糖化血红蛋白(HbA1c)等血液生化指标进行比较.[结果]430例老年糖尿病患者GAD-Ab、ICA、IAA和IA-2-Ab的阳性率分别为39.7%、28.5%、17.0%和16.4%,四项自身抗体全阴患者124例,占37.6%,与有一项以上阳性患者比较,其FPG、2hPBG和HbA1c水平无显著差异(P>0.05).[结论]四项联合检测对老年糖尿病患者的诊断及治疗方案的制定有重要意义.%[Objective] To explore the value of combined detection of glutamic acid decarboxylase antibody (GAD-Ab) , islet cell antibody(ICA) , insulin autoantibody(IAA) and protein-tyrosine phosphatase antibody (IA-2-Ab) in elderly patients with diabetes mellitus(DM). [Methods] Western blotting was used to detect serum GAD-Ab, ICA, IAA and IA-2-Ab in 430 elderly patients with DM and 40 healthy people. Biochemical indicators such as fasting plasma glucose (FPG) , postprandial 2h-blood glucose (PBG) and HbA1c were compared between patients with all 4 negative autoantibodies and patients with one or more positive autoantibod-ies. [Results]The positive rate of GAD-Ab, ICA, IAA and IA-2-Ab in 430 elderly patients with DM was 39. 7 %, 28. 5%, 17. 0% and 16. 4%, respectively. There were 124 patients(37. 6%) with all 4 negative autoantibodies. There was no difference in FPG, PBG and HbAlc between patients with all 4 negative autoantibodies and patients with one or more positive autoantibodies. [Conclusion] The combined detection of GAD-Ab, ICA, IAA and IA-2-Ab in serum have the important significance for the diagnosis and treatment protocol of elderly patients

  15. Stiff-Person Syndrome (United States)

    ... a blood test that measures the level of glutamic acid decarboxylase (GAD) antibodies in the blood. People with ... a blood test that measures the level of glutamic acid decarboxylase (GAD) antibodies in the blood. People with ...

  16. Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    CHAO Chen; HUANG Gan; LI Xia; YANG Lin; LIN Jian; JIN Ping; LUO Shuo-ming


    Background Glutamic acid decarboxylase antibody (GADA) and protein tyrosine phosphatase antibody (IA-2A) are two major autoantibodies,which exert important roles in the process of type 1 diabetes mellitus (T1D).Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.Methods Two hundreds and forty-seven Chinese newly diagnosed acute-onset T1D patients were consecutively recruited.GADA and IA-2A were detected at the time of diagnosis,one year later,3-5 years later after diagnosis during the follow-up; all the clinical data were recorded and analyzed as well.Results During the course of acute-onset T1D,the majority of patients remained stable for GADA or IA-2A,however,a few patients changed from positivity to negativity and fewer patients converted from negativity to positivity.The prevalence of GADA was 56.3% at diagnosis,decreasing to 50.5% one year later,and 43.3% 3-5 years later while the corresponding prevalence of IA-2A were 32.8%,31.0% and 23.3%,respectively.The median GADA titers were 0.0825 at diagnosis,declining to 0.0585 one year later and 0.0383 3-5 years later (P <0.001),while the corresponding median titers were 0.0016,0.0010,0.0014 for IA-2A,respectively.Fasting C-peptide (FCP) and postprandial C-peptide 2 hours (PCP2h)levels of GADA or IA-2A negativity persistence patients were higher than those of positivity persistence and negativity conversion patients (P <0.05) which indicated GADA or IA-2A negativity persistence T1D patients had a less loss of β cell function.Conclusion Our data suggest that repeated detection of GADA and IA-2A are necessary for differential diagnosis of autoimmune diabetes and the indirect prediction of the β cell function in Chinese patients.

  17. Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

    Directory of Open Access Journals (Sweden)

    Woods C Geoffrey


    Full Text Available Abstract Background Cerebral palsy (CP is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. Methods Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67, involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA. Results A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. Conclusions This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS, epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts. Table 4 GAD1 single nucleotide substitutions detected on mutation analysis and occurring in sequences submitted to NCBI SNP database and in the literature. This is not a definitive list, but includes those described at the time of the mutational analysis. *Nucleotide positions were not provided by Maestrini et al. [47]. Source SNP position in mRNA, from the translational start site (bp Gene position of SNP(bp Amino acid change (ALappalainen et al. (2002 A(-478Del Exon

  18. GAD65 antibodies among Greenland Inuit and its relation to glucose intolerance. (United States)

    Pedersen, Michael Lynge; Bjerregaard, Peter; Jørgensen, Marit Eika


    The aim of this study was to compare the prevalence of circulating Glutamin-Acid-decarboxylase 65 antibodies in a sample of Greenlanders (Inuit) with clinically verified diabetes with samples of participants from a population survey. The study population included participants with known diabetes from a population-based study (sample 1) and patients with clinically verified diabetes in Nuuk Greenland (sample 2). In addition, age- and gender-matched participants from the population study without known diabetes were categorized in groups with (1) normal glucose tolerance test, (2) with impaired fasting glycemia, (3) with impaired glucose tolerance and (4) with previously unknown diabetes based on oral glucose tolerance test and were enrolled in the study. Presence of circulating Glutamin-Acid-decarboxylase 65 antibodies were measured in all participants. A total of 484 persons were enrolled in the study. Six individuals had circulating Glutamin-Acid-decarboxylase 65 antibodies: four of them had known diabetes, one had impaired glucose tolerance and one normal glucose tolerance test. The prevalence of circulating Glutamin-Acid-decarboxylase 65 antibodies among Greenlanders with diabetes was 4.3 % and less than 1 % among Greenlanders without diabetes (p = 0.001). The prevalence of circulating Glutamin-Acid-decarboxylase 65 antibodies among Greenlanders with and without diabetes is relatively low in a global perspective in accordance with one former study among Inuit. Autoimmune diabetes seems to be uncommon in Greenland .

  19. GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota. (United States)

    Yunes, R A; Poluektova, E U; Dyachkova, M S; Klimina, K M; Kovtun, A S; Averina, O V; Orlova, V S; Danilenko, V N


    Gamma-amino butyric acid (GABA) is an active biogenic substance synthesized in plants, fungi, vertebrate animals and bacteria. Lactic acid bacteria are considered the main producers of GABA among bacteria. GABA-producing lactobacilli are isolated from food products such as cheese, yogurt, sourdough, etc. and are the source of bioactive properties assigned to those foods. The ability of human-derived lactobacilli and bifidobacteria to synthesize GABA remains poorly characterized. In this paper, we screened our collection of 135 human-derived Lactobacillus and Bifidobacterium strains for their ability to produce GABA from its precursor monosodium glutamate. Fifty eight strains were able to produce GABA. The most efficient GABA-producers were Bifidobacterium strains (up to 6 g/L). Time profiles of cell growth and GABA production as well as the influence of pyridoxal phosphate on GABA production were studied for L. plantarum 90sk, L. brevis 15f, B. adolescentis 150 and B. angulatum GT102. DNA of these strains was sequenced; the gadB and gadC genes were identified. The presence of these genes was analyzed in 14 metagenomes of healthy individuals. The genes were found in the following genera of bacteria: Bacteroidetes (Bacteroides, Parabacteroides, Alistipes, Odoribacter, Prevotella), Proteobacterium (Esherichia), Firmicutes (Enterococcus), Actinobacteria (Bifidobacterium). These data indicate that gad genes as well as the ability to produce GABA are widely distributed among lactobacilli and bifidobacteria (mainly in L. plantarum, L. brevis, B. adolescentis, B. angulatum, B. dentium) and other gut-derived bacterial species. Perhaps, GABA is involved in the interaction of gut microbiota with the macroorganism and the ability to synthesize GABA may be an important feature in the selection of bacterial strains - psychobiotics.

  20. Identification of γ-aminobutyric Acid producing Enterococcus faecium and Characterization of Its Glutamate Decarboxylase%产γ-氨基丁酸屎肠球菌的鉴定及其谷氨酸脱羧酶酶学性质

    Institute of Scientific and Technical Information of China (English)

    李云; 杨胜远; 杨韵晴; 黄荣城; 陈郁娜; 刘祥流


    目的: 鉴定1株产γ氨基丁酸(γ-aminobutyric acid,GABA)的乳酸菌HS3,并研究了其谷氨酸脱羧酶(Glutamate decarboxylase,GAD)粗酶酶学性质.方法:根据形态培养特征、生理生化特征和16S rDNA序列比对及系统发育分析对菌株HS3进行了鉴定.采用菌体细胞破碎后的粗酶液,研究了温度、pH和金属离子对酶活的影响.结果:菌株HS3的形态培养和生理生化特征符合肠球菌属(Enterococcus)特征,其16S rDNA序列与Enterococcus faecium(EU717962)16S rDNA序列同源性达99%,鉴定菌株HS3为屎肠球菌(Enterococcus faecium),菌株HS3 GAD最适作用温度为40 ℃,最适作用pH4.5.酶的热稳定较好,50℃处理4h,在pH3.5~6.0酶活基本稳定.Ca~(2+)对酶有激活作用,5mmol/L和50mmol/L浓度酶活分别提高了37.41%和17.43%.Ba~(2+)和Zn~(2+)在5mmol/L浓度时激活作用明显,而Mg~(2+)在5mmol/L浓度激活作用较好.结论:菌株HS3的GAD活力较高,稳定性较好,为生物合成GABA提供了新的微生物菌种资源.

  1. Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine. (United States)

    Su, Lingqia; Huang, Yan; Wu, Jing


    This report describes the optimization of recombinant Escherichia coli glutamate decarboxylase (GAD) production from engineered E. coli BL21(DE3) in a 3-L fermentor. Investigation of different induction strategies revealed that induction was optimal when the temperature was maintained at 30°C, the inducer (lactose) was fed at a rate of 0.2 g L(-1)h(-1), and protein expression was induced when the cell density (OD600) reached 50. Under these conditions, the GAD activity of 1273.8 U mL(-1) was achieved. Because GAD is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, the effect of supplementing the medium with pyridoxine hydrochloride (PN), a cheap and stable PLP precursor, on GAD production was also investigated. When the culture medium was supplemented with PN to a concentration of 2mM at the initiation of protein expression, and then again 10h later, the GAD activity reached 3193.4 U mL(-1), which represented the highest GAD production ever reported.

  2. CREB-Dependent Regulation of GAD65 Transcription by BDNF/TrkB in Cortical Interneurons. (United States)

    Sánchez-Huertas, Carlos; Rico, Beatriz


    In the cerebral cortex, the functional output of projection neurons is fine-tuned by inhibitory neurons present in the network, which use γ-aminobutyric acid (GABA) as their main neurotransmitter. Previous studies have suggested that the expression levels of the rate-limiting GABA synthetic enzyme, GAD65, depend on brain derived neurotrophic factor (BDNF)/TrkB activation. However, the molecular mechanisms by which this neurotrophic factor and its receptor controls GABA synthesis are still unknown. Here, we show a direct regulation of the GAD65 gene by BDNF-TrkB signaling via CREB in cortical interneurons. Conditional ablation of TrkB in cortical interneurons causes a cell-autonomous decrease in the synaptically enriched GAD65 protein and its transcripts levels, suggesting that transcriptional regulation of the GAD65 gene is altered. Dissection of the intracellular pathway that underlies this process revealed that BDNF/TrkB signaling controls the transcription of GAD65 in a Ras-ERK-CREB-dependent manner. Our study reveals a novel molecular mechanism through which BDNF/TrkB signaling may modulate the maturation and function of cortical inhibitory circuits.

  3. Cultural-based biases of the GAD-7. (United States)

    Parkerson, Holly A; Thibodeau, Michel A; Brandt, Charles P; Zvolensky, Michael J; Asmundson, Gordon J G


    The GAD-7 is a popular measure of generalized anxiety disorder (GAD) symptoms that has been used across many cultural groups. Existing evidence demonstrates that the prevalence of GAD varies across self-identified ethnic/cultural groups, a phenomenon that some researchers attribute to cross-cultural measurement error rather than to actual differences in rates of GAD. Nonetheless, the effect of culture on factor structure and response patterns to the GAD-7 have not been examined and could result over- or under-estimated GAD-7 scores across different cultural groups. The current investigation assessed the factor structure of the GAD-7 in White/Caucasian, Hispanic, and Black/African American undergraduates and tested for cultural-based biases. A modified one-factor model exhibited good fit across subsamples. Results revealed that Black/African American participants with high GAD symptoms scored lower on the GAD-7 than other participants with similar GAD symptoms. Results highlight the need for culturally sensitive GAD screening tools.

  4. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

    DEFF Research Database (Denmark)

    Walls, Anne Byriel; Eyjolfsson, Elvar M.; Smeland, Olav B.


    was further investigated in GAD65 knockout and wild-type mice using [1,2-(13)C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex...... glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice....

  5. A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

    Directory of Open Access Journals (Sweden)

    Vogel Constantin


    Full Text Available Abstract Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD is still missing. Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult. Results We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8 was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED and was used to systematically analyze functionally and structurally relevant

  6. Role of glutamate decarboxylase-like protein 1 (GADL1) in taurine biosynthesis. (United States)

    Liu, Pingyang; Ge, Xiaomei; Ding, Haizhen; Jiang, Honglin; Christensen, Bruce M; Li, Jianyong


    This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

  7. Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad-substrate-specificity 2-oxo-acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483. (United States)

    Bolat, Irina; Romagnoli, Gabriele; Zhu, Feibai; Pronk, Jack T; Daran, Jean-Marc


    The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80, which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc-like and one Seub-like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing 'trans' regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.

  8. Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. (United States)

    Phelps, Edward A; Cianciaruso, Chiara; Michael, Iacovos P; Pasquier, Miriella; Kanaani, Jamil; Nano, Rita; Lavallard, Vanessa; Billestrup, Nils; Hubbell, Jeffrey A; Baekkeskov, Steinunn


    Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

  9. A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  10. A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer. (United States)

    Ippolito, Joseph E; Piwnica-Worms, David


    Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  11. Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its γ-aminobutyric acid biosynthesis ability in milk. (United States)

    Wu, Qinglong; Law, Yee-Song; Shah, Nagendra P


    Most high γ-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.

  12. Cloning and Prokaryotic Expressing of Glutamate Decarboxylase Gene from Lactobacillus plantarum%植物乳杆菌谷氨酸脱羧酶基因的克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    时粲; 刘昭明; 黎娅; 易弋; 伍时华


    Based on the sequence of Lactobacillus plantarum WCFS1 published in GenBank, primers were designed and the glutamate decarboxylase encoding gene gadB from L. plantarum QL-14 was amplified by PCR technology. Then the target gene was expressed in Escherichia coli BL21 through fusion expression vector pGEX-4T-3, and the expression condition was optimized and target protein was purified. The results showed that the ORF of gadB gene was 1 407 bp and encoded 469 amino acids, 99.6% homology with L. plantarum WCFS1. After optimizing the inducing condition, the molecular weight and pI of purified GAD protein reached to 53.6 ku and 5.58 respectively, with an activity of 9.9 U/mg.%根据GenBank中植物乳杆菌(Lactobacillus plantarum)基因组序列设计引物,利用PCR技术扩增了植物乳杆菌QL-14的谷氨酸脱羧酶编码基因gadB,克隆至表达载体pGEX-4T-3,转化大肠杆菌(Es-cherichia coli)后进行原核表达,对表达条件进行了优化并对表达蛋白质进了纯化。结果表明,扩增目的gadB基因的开放阅读框(ORF)全长1407 bp,编码469个氨基酸,氨基酸序列与植物乳杆菌WCFS1同源性为99.6%。通过优化诱导表达条件,得到纯化蛋白质GAD大小为53.6 ku,等电点为5.58,比活力为9.9 U/mg。

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

    Kuo, D J; Jordan, F


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

  14. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity. (United States)

    Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo


    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.

  15. Corticotropin-releasing hormone (CRH)-containing neurons in the immature rat hippocampal formation: light and electron microscopic features and colocalization with glutamate decarboxylase and parvalbumin. (United States)

    Yan, X X; Toth, Z; Schultz, L; Ribak, C E; Baram, T Z


    Corticotropin-releasing hormone (CRH) excites hippocampal neurons and induces death of selected CA3 pyramidal cells in immature rats. These actions of CRH require activation of specific receptors that are abundant in CA3 during early postnatal development. Given the dramatic effects of CRH on hippocampal neurons and the absence of CRH-containing afferents to this region, we hypothesized that a significant population of CRHergic neurons exists in developing rat hippocampus. This study defined and characterized hippocampal CRH-containing cells by using immunocytochemistry, ultrastructural examination, and colocalization with gamma-aminobutyric acid (GABA)-synthesizing enzyme and calcium-binding proteins. Numerous, large CRH-immunoreactive (ir) neurons were demonstrated in CA3 strata pyramidale and oriens, fewer were observed in the corresponding layers of CA1, and smaller CRH-ir cells were found in stratum lacunosum-moleculare of Ammon's horn. In the dentate gyrus, CRH-ir somata resided in the granule cell layer and hilus. Ultrastructurally, CRH-ir neurons had aspiny dendrites and were postsynaptic to both asymmetric and symmetric synapses. CRH-ir axon terminals formed axosomatic and axodendritic symmetric synapses with pyramidal and granule cells. Other CRH-ir terminals synapsed on axon initial segments of principal neurons. Most CRH-ir neurons were coimmunolabeled for glutamate decarboxylase (GAD)-65 and GAD-67 and the majority also contained parvalbumin, but none were labeled for calbindin. These results confirm the identity of hippocampal CRH-ir cells as GABAergic interneurons. Further, a subpopulation of neurons immunoreactive for both CRH and parvalbumin and located within and adjacent to the principal cell layers consists of basket and chandelier cells. Thus, axon terminals of CRH-ir interneurons are strategically positioned to influence the excitability of the principal hippocampal neurons via release of both CRH and GABA.

  16. Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration. (United States)

    Stanley, Dirk P; Shetty, Ashok K


    Increased excitability of principal excitatory neurons is one of the hallmarks of aging in the hippocampus, signifying a diminution in the number and/or function of inhibitory interneurons with aging. To elucidate this, we performed comprehensive GABA-ergic interneuron cell counts in all layers of the dentate gyrus and the CA1 and CA3 subfields, using serial sections from adult, middle-aged and aged Fischer 344 rats. Sections were immunostained for glutamate decarboxylase-67 (GAD-67, a synthesizing enzyme of GABA) and GAD-67 immunopositive interneurons were counted using an unbiased cell counting method, the optical fractionator. Substantial declines in the absolute number of GAD-67 immunopositive interneurons were found in all hippocampal layers/subfields of middle-aged and aged animals, in comparison with the adult animals. However, the counts were comparable between the middle-aged and aged groups for all regions. Interestingly, determination of the absolute number of interneurons using neuron-specific nuclear antigen (NeuN) expression in the strata oriens and radiatum of CA1 and CA3 subfields revealed an analogous number of interneurons across the three age groups. Furthermore, the ratio of GAD-67 immunopositive and NeuN positive interneurons decreased from adult age to middle age but remained relatively static between middle age and old age. Collectively, the results underscore that aging in the hippocampus is associated with wide-ranging decreases in the number of GAD-67 immunopositive interneurons and most of the age-related changes in GAD-67 immunopositive interneuron numbers transpire by middle age. Additionally, this study provides novel evidence that age-related reductions in hippocampal GAD-67 immunopositive interneuron numbers are due to loss of GAD-67 expression in interneurons rather than interneuron degeneration.

  17. Intracerebroventricular kainic acid administration to neonatal rats alters interneuron development in the hippocampus. (United States)

    Dong, Hongxin; Csernansky, Cynthia A; Chu, Yunxiang; Csernansky, John G


    The effects of neonatal exposure to excitotoxins on the development of interneurons have not been well characterized, but may be relevant to the pathogenesis of neuropsychiatric disorders. In this study, the excitotoxin, kainic acid (KA) was administered to rats at postnatal day 7 (P7) by intracerebroventricular (i.c.v.) infusion. At P14, P25, P40 and P60, Nissl staining and immunohistochemical studies with the interneuron markers, glutamic acid decarboxylase (GAD-67), calbindin-D28k (CB) and parvalbumin (PV) were performed in the hippocampus. In control animals, the total number of interneurons, as well as the number of interneurons stained with GAD-67, CB and PV, was nearly constant from P14 through P60. In KA-treated rats, Nissl staining, GAD-67 staining, and CB staining revealed a progressive decline in the overall number of interneurons in the CA1 and CA3 subfields from P14 to P60. In contrast, PV staining in KA-treated rats showed initial decreases in the number of interneurons in the CA1 and CA3 subfields at P14 followed by increases that approached control levels by P60. These results suggest that, in general, early exposure to the excitotoxin KA decreases the number of hippocampal interneurons, but has a more variable effect on the specific population of interneurons labeled by PV. The functional impact of these changes may be relevant to the pathogenesis of neuropsychiatric disorders, such as schizophrenia.

  18. Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies

    DEFF Research Database (Denmark)

    Pörksen, Sven; Laborie, Lene Bjerke; Nielsen, Lotte


    BACKGROUND: To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (...

  19. Generalized Anxiety Disorder (GAD): When Worry Gets Out of Control (United States)

    ... health, money, or family problems. But people with generalized anxiety disorder (GAD) feel extremely worried or feel nervous about ... I find more information? To learn more about generalized anxiety disorder, visit: MedlinePlus (National Library of Medicine) http: / / medlineplus. ...

  20. Comparison of Measurements of Autoantibodies to Glutamic Acid Decarboxylase and Islet Antigen-2 in Whole Blood Eluates from Dried Blood Spots Using the RSR-Enzyme Linked Immunosorbent Assay Kits and In-House Radioimmunoassays

    Directory of Open Access Journals (Sweden)

    Anders Persson


    Full Text Available To evaluate the performance of dried blood spots (DBSs with subsequent analyses of glutamic acid decarboxylase (GADA and islet antigen-2 (IA-2A with the RSR-ELISAs, we selected 80 children newly diagnosed with type 1 diabetes and 120 healthy women. DBSs from patients and controls were used for RSR-ELISAs while patients samples were analysed also with in-house RIAs. The RSR-ELISA-GADA performed well with a specificity of 100%, albeit sensitivity (46% was lower compared to in RIA (56%; P=.008. No prozone effect was observed after dilution of discrepant samples. RSR-ELISA-IA-2A achieved specificity of 69% and sensitivity was lower (59% compared with RIA (66%; P<.001. Negative or low positive patients and control samples in the RSR-ELISA-IA-2A increased after dilution. Eluates from DBS can readily be used to analyse GADA with the RSR-ELISA, even if low levels of autoantibodies were not detected. Some factor could disturb RSR-ELISA-IA-2A analyses.

  1. [Influence of exogenous gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid contents in roots of melon seedling under hypoxia stress]. (United States)

    Wang, Chun-Yan; Li, Jing-Rui; Xia, Qing-Ping; Wu, Xiao-Lei; Gao, Hong-Bo


    This paper investigated the influence of gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid content under hypoxia stress by accurately controlling the level of dissolved oxygen in hydroponics, using the roots of melon 'Xiyu 1' seedlings as the test material. The results showed that compared with the control, the growth of roots was inhibited seriously under hypoxia stress. Meanwhile, the hypoxia-treated roots had significantly higher activities of glutamate decarboxylase (GAD), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as the contents of GABA, pyruvic acid, alanine (Ala) and aspartic acid (Asp). But the contents of glutamic acid (Glu) and alpha-keto glutaric acid in roots under hypoxia stress was obviously lower than those of the control. Exogenous treatment with GABA alleviated the inhibition effect of hypoxia stress on root growth, which was accompanied by an increase in the contents of endogenous GABA, Glu, alpha-keto glutaric acid and Asp. Furthermore, under hypoxia stress, the activities of GAD, GDH, GOGAT, GS, ALT, AST as well as the contents of pyruvic acid and Ala significantly decreased in roots treated with GABA. However, adding GABA and viny-gamma-aminobutyric acid (VGB) reduced the alleviation effect of GABA on melon seedlings under hypoxia stress. The results suggested that absorption of GABA by roots could alleviate the injury of hypoxia stress to melon seedlings. This meant that GABA treatment allows the normal physiological metabolism under hypoxia by inhibiting the GAD activity through feedback and maintaining higher Glu content as well as the bal- ance of carbon and nitrogen.

  2. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase (United States)

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian


    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  3. Characterization of arginine decarboxylase from Dianthus caryophyllus. (United States)

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


    Arginine decarboxylase (ADC, EC 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%.

  4. New insights into {gamma}-aminobutyric acid catabolism: Evidence for {gamma}-hydroxybutyric acid and polyhydroxybutyrate synthesis in Saccharomyces cerevisiae. (United States)

    Bach, Benoît; Meudec, Emmanuelle; Lepoutre, Jean-Paul; Rossignol, Tristan; Blondin, Bruno; Dequin, Sylvie; Camarasa, Carole


    The gamma-aminobutyrate (GABA) shunt, an alternative route for the conversion of alpha-ketoglutarate to succinate, involves the glutamate decarboxylase Gad1p, the GABA transaminase Uga1p and the succinate semialdehyde dehydrogenase Uga2p. This pathway has been extensively described in plants and animals, but its function in yeast remains unclear. We show that the flux through Gad1p is insignificant during fermentation in rich sugar-containing medium, excluding a role for this pathway in redox homeostasis under anaerobic conditions or sugar stress. However, we found that up to 4 g of exogenous GABA/liter was efficiently consumed by yeast. We studied the fate of this consumed GABA. Most was converted into succinate, with a reaction yield of 0.7 mol/mol. We also showed that a large proportion of GABA was stored within cells, indicating a possible role for this molecule in stress tolerance mechanisms or nitrogen storage. Furthermore, based on enzymatic and metabolic evidence, we identified an alternative route for GABA catabolism, involving the reduction of succinate-semialdehyde into gamma-hydroxybutyric acid and the polymerization of gamma-hydroxybutyric acid to form poly-(3-hydroxybutyric acid-co-4-hydroxybutyric acid). This study provides the first demonstration of a native route for the formation of this polymer in yeast. Our findings shed new light on the GABA pathway and open up new opportunities for industrial applications.

  5. Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor (United States)

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


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

  6. Glutamine, glutamate, and arginine-based acid resistance in Lactobacillus reuteri. (United States)

    Teixeira, Januana S; Seeras, Arisha; Sanchez-Maldonado, Alma Fernanda; Zhang, Chonggang; Su, Marcia Shu-Wei; Gänzle, Michael G


    This study aimed to determine whether glutamine deamidation improves acid resistance of Lactobacillus reuteri, and to assess whether arginine, glutamine, and glutamate-mediated acid resistance are redundant or complementary mechanisms of acid resistance. Three putative glutaminase genes, gls1, gls2, and gls3, were identified in L. reuteri 100-23. All three genes were expressed during growth in mMRS and wheat sourdough. L. reuteri consistently over-expressed gls3 and the glutamate decarboxylase gadB. L. reuteri 100-23ΔgadB over-expressed gls3 and the arginine deiminase gene adi. Analysis of the survival of L. reuteri in acidic conditions revealed that arginine conversion is effective at pH of 3.5 while glutamine or glutamate conversion were effective at pH of 2.5. Arginine conversion increased the pHin but not ΔΨ; glutamate decarboxylation had only a minor effect on the pHin but increased the ΔΨ. This study demonstrates that glutamine deamidation increases the acid resistance of L. reuteri independent of glutamate decarboxylase activity. Arginine and glutamine/glutamate conversions confer resistance to lactate at pH of 3.5 and phosphate at pH of 2.5, respectively. Knowledge of L. reuteri's acid resistance improves the understanding of the adaptation of L. reuteri to intestinal ecosystems, and facilitates the selection of probiotic and starter cultures.

  7. Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-gamma-glutamyl hydrazone in the developing rat. (United States)

    Massieu, L; Rivera, A; Tapia, R


    We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5'-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B6 depletion, but alterations of other B6-dependent biochemical pathways cannot be discarded.

  8. Impact of Precooling and Controlled-Atmosphere Storage on γ-Aminobutyric Acid (GABA) Accumulation in Longan (Dimocarpus longan Lour.) Fruit. (United States)

    Zhou, Molin; Ndeurumio, Kessy H; Zhao, Lei; Hu, Zhuoyan


    Longan (Dimocarpus longan Lour.) fruit cultivars 'Chuliang' and 'Shixia' were analyzed for γ-aminobutyric acid (GABA) accumulation after precooling and in controlled-atmosphere storage. Fruit were exposed to 5% O2 plus 3%, 5%, or 10% CO2 at 4 °C, and GABA and associated enzymes, aril firmness, and pericarp color were measured. Aril softening and pericarp browning were delayed by 5% CO2 + 5% O2. GABA concentrations and glutamate decarboxylase (GAD; EC activities declined during storage at the higher-CO2 treatments. However, GABA aminotransferase (GABA-T; EC activities in elevated CO2-treated fruit fluctuated during storage. GABA concentrations increased after precooling treatments. GAD activity and GABA-T activity were different between cultivars after precooling. GABA concentrations in fruit increased after 3 days of 10% CO2 + 5% O2 treatment and then declined as storage time increased. GABA accumulation was associated with stimulation of GAD activity rather than inhibition of GABA-T activity.

  9. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a. (United States)

    Vanderslice, P; Copeland, W C; Robertus, J D


    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  10. 牛奶中分离的乳酸菌GGMCC1306中谷氨酸脱羧酶的分离纯化及酶学性质研究%Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

    Institute of Scientific and Technical Information of China (English)

    黄俊; 梅乐和; 盛清; 姚善泾; 林东强


    A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%-90% saturation (NH4)2SO4 fractional precipitation,Q sepharose FF anion-exchange chromatography,sephacryl S-200 gel filtration,and resource Q anion-exchange chromatography.Using this protocol,the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purificaton fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃,respecively.The purified GAD had a half-life of 50minat 45℃ and the Km value of the enzyme from Lineweaver-Burk polt was found to be 8.22.5'-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.

  11. Transcriptome and metabolome analyses of sugar and organic acid metabolism in Ponkan (Citrus reticulata) fruit during fruit maturation. (United States)

    Lin, Qiong; Wang, Chengyang; Dong, Wencheng; Jiang, Qing; Wang, Dengliang; Li, Shaojia; Chen, Ming; Liu, Chunrong; Sun, Chongde; Chen, Kunsong


    Ponkan (Citrus reticulata Blanco cv. Ponkan) is an important mandarin citrus in China. However, the low ratio of sugars to organic acids makes it less acceptable for consumers. In this work, three stages (S120, early development stage; S195, commercial harvest stage; S205, delayed harvest stage) of Ponkan fruit were selected for study. Among 28 primary metabolites analyzed in fruit, sugars increased while organic acids in general decreased. RNA-Seq analysis was carried out and 19,504 genes were matched to the Citrus clementina genome, with 85 up-regulated and 59 down-regulated genes identified during fruit maturation. A sucrose phosphate synthase (SPS) gene was included in the up-regulated group, and this was supported by the transcript ratio distribution. Expression of two asparagine transferases (AST), and a specific ATP-citrate lyase (ACL) and glutamate decarboxylase (GAD) members increased during fruit maturation. It is suggested that SPS, AST, ACL and GAD coordinately contribute to sugar accumulation and organic acid degradation during Ponkan fruit maturation. Both the glycolysis pathway and TCA cycle were accelerated during later maturation, indicating the flux change from sucrose metabolism to organic acid metabolism was enhanced, with citrate degradation occurring mainly through the gamma-aminobutyric acid (GABA) and acetyl-CoA pathways.

  12. Glutaminsyre-decarboxylase-antistoffer og diabetes

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, Thomas


    The 1999 WHO classification delineates immune mediated type 1 diabetes from other types of diabetes by the presence of auto-antibodies against beta-cell constituents. The GAD65 auto-antibody test is the method of first choice because it has the highest sensitivity, specificity and positive...... predictive value and is the most standardized and well-characterized type 1 diabetes related auto-antibody analysis. It is recommended that demonstration of GAD auto-antibodies leads to diagnosis, classification or re-classification of diabetes patients as immune mediated type 1 diabetes. Udgivelsesdato...

  13. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes. (United States)

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


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

  14. Conformational Stabilization of Rat S-Adenosylmethionine Decarboxylase by Putrescine


    和田, 牧子; 白幡, 晶


    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using m...

  15. Decline in titers of anti-idiotypic antibodies specific to autoantibodies to GAD65 (GAD65Ab precedes development of GAD65Ab and type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Helena Elding Larsson

    Full Text Available The humoral Idiotypic Network consisting of antibodies and their anti-idiotypic antibodies (anti-Id can be temporarily upset by antigen exposure. In the healthy immune response the original equilibrium is eventually restored through counter-regulatory mechanisms. In certain autoimmune diseases however, autoantibody levels exceed those of their respective anti-Id, indicating a permanent disturbance in the respective humoral Idiotypic Network. We investigated anti-Id directed to a major Type 1 diabetes (T1D-associated autoantibody (GAD65Ab in two independent cohorts during progression to disease. Samples taken from participants of the Natural History Study showed significantly lower anti-Id levels in individuals that later progressed to T1D compared to non-progressors (anti-Id antibody index of 0.06 vs. 0.08, respectively, p = 0.02. We also observed a significant inverse correlation between anti-Id levels and age at sampling, but only in progressors (p = 0.014. Finally, anti-Id levels in progressors showed a significant decline during progression as compared to longitudinal anti-Id levels in non-progressors (median rate of change: -0.0004 vs. +0.0004, respectively, p = 0.003, suggesting a loss of anti-Id during progression. Our analysis of the Diabetes Prediction in Skåne cohort showed that early in life (age 2 individuals at risk have anti-Id levels indistinguishable from those in healthy controls, indicating that low anti-Id levels are not an innate characteristic of the immune response in individuals at risk. Notably, anti-Id levels declined significantly in individuals that later developed GAD65Ab suggesting that the decline in anti-Id levels precedes the emergence of GAD65Ab (median rate of change: -0.005 compared to matched controls (median rate of change: +0.001 (p = 0.0016. We conclude that while anti-Id are present early in life, their levels decrease prior to the appearance of GAD65Ab and to the development of T1D.

  16. 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


    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.

  17. Enlargement of Axo-Somatic Contacts Formed by GAD-Immunoreactive Axon Terminals onto Layer V Pyramidal Neurons in the Medial Prefrontal Cortex of Adolescent Female Mice Is Associated with Suppression of Food Restriction-Evoked Hyperactivity and Resilience to Activity-Based Anorexia. (United States)

    Chen, Yi-Wen; Wable, Gauri Satish; Chowdhury, Tara Gunkali; Aoki, Chiye


    Many, but not all, adolescent female mice that are exposed to a running wheel while food restricted (FR) become excessive wheel runners, choosing to run even during the hours of food availability, to the point of death. This phenomenon is called activity-based anorexia (ABA). We used electron microscopic immunocytochemistry to ask whether individual differences in ABA resilience may correlate with the lengths of axo-somatic contacts made by GABAergic axon terminals onto layer 5 pyramidal neurons (L5P) in the prefrontal cortex. Contact lengths were, on average, 40% greater for the ABA-induced mice, relative to controls. Correspondingly, the proportion of L5P perikaryal plasma membrane contacted by GABAergic terminals was 45% greater for the ABA mice. Contact lengths in the anterior cingulate cortex correlated negatively and strongly with the overall wheel activity after FR (R = -0.87, P < 0.01), whereas those in the prelimbic cortex correlated negatively with wheel running specifically during the hours of food availability of the FR days (R = -0.84, P < 0.05). These negative correlations support the idea that increases in the glutamic acid decarboxylase (GAD) terminal contact lengths onto L5P contribute toward ABA resilience through suppression of wheel running, a behavior that is intrinsically rewarding and helpful for foraging but maladaptive within a cage.

  18. Retina maturation following administration of thyroxine in developing rats: effects on polyamine metabolism and glutamate decarboxylase. (United States)

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


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


    Institute of Scientific and Technical Information of China (English)

    陈鹏; 李金莲


    Objective To observe the association of vesicular glutamate transporter of type Ⅰ(VGluT1)-like immunoreactive(LI),the differentiation-associated Na+-dependent inorganic phosphate cotransporter(DNPI)-LI and glutamic acid decarboxylase(GAD)-LI terminals with GABAA receptor α3 subunit(GABAARα3)-LI neurons in mesencephalic trigeminal nucleus of the rat. Methods Triple-immunofluorescence histochemical staining technique and confocal laser-scanning microscopy were used. Results A large number of neuronal cell bodies showed GABAA Rα3-LI immunoreactivity at all rostrocaudal levels of the Vme,and most of GABAARα3-LI cells were large (25-50μm) pseudounipolar neurons.The dense VGluT1-LI,DNPL-LI and GAD-LI terminals distributed widely in Vme,some VGluT1/DNPI-LI and GAD-LI terminals surrounded the somata of the GABAARα3-LI Vme neurons,and made close contacts with them.Conclusion Proprioceptive sensory signals from the orofacial region might be modulated at the level of the primary afferent cell bodies in the Vme both by glutamatergic- and GABAergic-axonal terminals from other brain areas,and the effect of GABAergic-terminals might be mediated by post-synaptical GABAA receptors.%目的观察大鼠三叉神经中脑核(Vme)内囊泡膜谷氨酸转运体(VGluT1和DNPI)样和谷氨酸脱羧酶(GAD)样阳性终末与GABAA受体α3亚单位(GABAARα3)样阳性神经元之间的联系. 方法免疫荧光组织化学三重染色技术,在激光共聚焦显微镜下观察. 结果在Vme吻尾方向上,有大量的神经元胞体呈GABAARα3样免疫阳性,这些神经元多为大的假单极神经元(直径为25~50μm).VGluT1样、DNPI样和GAD样免疫阳性终末密集分布于Vme内,一些VGluT1/DNPI样和GAD样阳性终末包绕在GABAARα3样阳性Vme神经元胞体周围,并与之形成密切接触. 结论 Vme神经元在介导口面部本体感觉信息的传递过程中,可能同时接受中枢其他来源的谷氨酸能和GABA能终末的调控,其中GABA能终末的

  20. Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies

    Directory of Open Access Journals (Sweden)

    de Beaufort Carine


    Full Text Available Abstract Background To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative for pancreatic autoantibodies [islet cell autoantibodies(ICA, glutamic acid decarboxylase antibodies (GADA and insulinoma-associated antigen-2 antibodies (IA-2A]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes. Materials and methods In 261 newly diagnosed children with type 1 diabetes, we measured residual β-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation. Results Twenty-four patients (9.1% tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual β-cell function (P = 0.002, lower blood glucose (P = 0.004, received less insulin (P = 0.05 and had lower HbA1c (P = 0.02 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8 by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide. Conclusion GAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.

  1. Optimization ofγ-aminobutyric Preparation by Recombinant Glutamate Decarboxylase%重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化

    Institute of Scientific and Technical Information of China (English)

    黄燕; 宿玲恰; 吴敬


    谷氨酸脱羧酶,一种磷酸吡哆醛(PLP)依赖性酶,能专一、不可逆地催化L-谷氨酸脱羧得到γ-氨基丁酸(GABA)。构建了产Lactobacillus brevisWJH3谷氨酸脱羧酶重组大肠杆菌E.coliBL21(DE3)/pET-24a-gad,以此作为菌种进行摇瓶发酵诱导培养,发酵过程中一次性添加0.05mmol/LPLP培养24h,破壁上清酶活达81.7U/mL,是不添加PLP对照酶活的1.8倍。对酶转化L-谷氨酸钠生成GABA反应条件进行了优化,结果表明,在转化体系不添加PLP的情况下,底物谷氨酸钠浓度为250g/L,反应初始pH5.0,温度37℃,加酶量60U/g底物,转速200r/min,在此条件下反应18h,GABA转化率达到100%,为γ-氨基丁酸的工业化生产奠定基础。%Glutamate decarboxylase(GAD),a pyridoxal 5'-phosphate(PLP)-dependent enzyme,irreversibly catalyzes the decarboxylation of L-glutamate to be the valuable food additive γ-aminobutyric acid(GABA). In this study,a recombinant Escherichia coli BL21(DE3)/pET-24a-gad producing Lactobacillus brevis WJH3 GAD was constructed as strain in the flask culturing of fermentation and induction. The activity of GAD produced in the supernatant of culturing for 24 h medium supplemented one-time with 0.05 mmol/L PLP was 81.7 U/mL,and this was 1.8-fold of that without PLP supplementation. Furthermore,the condition for GABA preparation by enzymatic conversion was optimized;under the condition of 250 g/L monosodium glutamate(MSG),pH5.0,37℃,60 U GAD per gram substrate incubated for 18 hours,and rotation rate 200 r/min,100% of the MSG was transformed into GABA. These results establish the utility of PLP supplementation and lay the foundation for large-scale enzymatic production of GABA.

  2. Evaluation of improved γ-aminobutyric acid production in yogurt using Lactobacillus plantarum NDC75017. (United States)

    Shan, Y; Man, C X; Han, X; Li, L; Guo, Y; Deng, Y; Li, T; Zhang, L W; Jiang, Y J


    Most γ-aminobutyric acid (GABA)-producing microorganisms are lactic acid bacteria (LAB), but the yield of GABA is limited in most of these GABA-producing strains. In this study, the production of GABA was carried out by using Lactobacillus plantarum NDC75017, a strain screened from traditional fermented dairy products in China. Concentrations of substrate (l-monosodium glutamate, L-MSG) and coenzyme (pyridoxal-5-phosphate, PLP) of glutamate decarboxylase (GAD) and culture temperature were investigated to evaluate their effects on GABA yield of Lb. plantarum NDC75017. The results indicated that GABA production was related to GAD activity and biomass of Lb. plantarum NDC75017. Response surface methodology was used to optimize conditions of GABA production. The optimal factors for GABA production were L-MSG at 80 mM, PLP at 18 μM, and a culture temperature of 36 °C. Under these conditions, production of GABA was maximized at 314.56 mg/100 g. Addition of Lb. plantarum NDC75017 to a commercial starter culture led to higher GABA production in fermented yogurt. Flavor and texture of the prepared yogurt and the control yogurt did not differ significantly. Thus, Lb. plantarum NDC75017 has good potential for manufacture of GABA-enriched fermented milk products.

  3. 糖尿病患者 GAD-Ab、IA2-Ab 和 ICA 联合检测的临床意义%Clinical significance of the combined detection of GAD-Ab, IA2-Ab and ICA in patients with diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    周厚清; 吴行贵


    目的:探讨抗谷氨酸脱羧酶抗体(GAD-Ab)、抗酪氨酸磷酸酶抗体(IA2-Ab)和抗胰岛细胞抗体(ICA)联合检测在糖尿病中的临床意义。方法选择糖尿病患者136例,其中1型糖尿病54例、2型糖尿病82例,同时选择健康对照者(正常对照组)65名,采用酶联免疫吸附试验(ELISA)检测 GAD-Ab 和 IA2-Ab,并采用间接免疫荧光法检测 ICA。结果1型糖尿病组 ICA、GAD-Ab 和 IA2-Ab 的阳性率分别为22.2%、61.1%和11.1%,明显高于2型糖尿病组(2.4%、4.9%、1.2%)及正常对照组(0%、0%、0%)(P 均<0.05)。1型糖尿病组 GAD-Ab阳性率明显高于 ICA 和 IA2-Ab(P <0.001)。 GAD-Ab 诊断1型糖尿病的敏感性和诊断符合率均高于 ICA 和IA2-Ab(P <0.05);GAD-Ab +ICA、GAD-Ab +IA2-Ab、ICA +GAD-Ab +IA2-Ab 联合检测的敏感性和诊断符合率亦均高于 ICA 和 IA2-Ab 单项检测(P <0.05)。结论 GAD-Ab、IA2-Ab 和 ICA 联合检测对糖尿病的诊断分型具有一定的意义。%Objective To investigate the clinical significance of the combined detection of anti-glutamic acid decarboxylase antibody (GAD-Ab), anti-protein tyrosine phosphatase antibody (IA2-Ab) and anti-islet cell antibody (ICA) in the diagnosis of diabetes mellitus.Methods A total of 136 patients with diabetes mellitus were enrolled, including 54 cases of type 1 diabetes mellitus and 82 cases of type 2 diabetes mellitus, and 65 healthy controls were enrolled as healthy control group.GAD-Ab and IA2-Ab were determined by enzyme-linked immunosorbent assay (ELISA), and ICA was determined by indirect immunofluorescence assay.Results The positive rates of ICA, GAD-Ab and IA2-Ab in patients with type 1 diabetes mellitus(22.2%, 61.1% and 11.1%) were significantly higher than those in patients with type 2 diabetes mellitus (2.4%, 4.9% and 1.2%) and control group (0%, 0% and

  4. Isolation and bioinformatics analysis of a glutamate decarboxylase gene inDendrobium officinale%铁皮石斛谷氨酸脱羧酶基因的分离与生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    张岗; 胡本祥; 李依民; 张大为; 郭顺星


    目的分离珍稀濒危兰科药用铁皮石斛谷氨酸脱羧酶(GAD)基因并进行生物信息学和表达分析。方法采用 RT-PCR和 RACE技术获基因 cDNA全长;利用生物信息学软件分析蛋白理化性质、结构域和三维建模等分子特性;用 DNASTAR 6.0和 MEGA 4.0分别进行氨基酸多序列比对和进化树分析;借助实时定量 PCR检测基因表达。结果分离到DoGAD基因,cDNA全长1795 bp,编码一条由498个氨基酸组成的多肽,分子量55.90 kD,等电点5.32;DoGAD蛋白不含跨膜域或信号肽,具有谷氨酸脱羧酶和磷酸吡哆醛依赖的脱羧酶结构域(17-443、37-381);DoGAD与植物 GADs蛋白一致性为69.5%~78.8%,隶属于 GADs分子进化树的植物类群;DoGAD转录本在石斛叶和茎中相对表达量较高,分别为根中的5.16和3.92倍。结论成功克隆得到铁皮石斛谷氨酸脱羧酶基因全长, DoGAD 的表达特征暗示其可能在铁皮石斛叶和茎中发挥重要的调控作用。%ObjectiveThis study is aimed to isolate and characterize a glutamate decarboxylase (GAD) geneDoGAD fromDendrobium officinale, a rare endangered medicinal orchid species. MethodsRT-PCR and RACE technologies were used for gene isolation. The physiochemical properties, conserved domains and three dimensional structure of the deduced DoGAD protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0, respectively. Real time quantitative PCR was used for gene expression analysis.Results The full-length cDNA ofDoGAD, with 1795 bp in size, was deduced to encode a 498-aa protein with molecular weight of 55.90 kD and isoelectric point of 5.32. The deduced DoGAD protein, without transmembrane or signal peptide residues, contained glutamate decarboxylase and pyridoxal phosphate dependant decarboxylase domains (17-443, 37-381). DoGAD had high identities (69.5% - 78

  5. Diagnostic Validity of the Generalized Anxiety Disorder - 7 (GAD-7 among Pregnant Women.

    Directory of Open Access Journals (Sweden)

    Qiu-Yue Zhong

    Full Text Available Generalized anxiety disorder (GAD during pregnancy is associated with several adverse maternal and perinatal outcomes. A reliable and valid screening tool for GAD should lead to earlier detection and treatment. Among pregnant Peruvian women, a brief screening tool, the GAD-7, has not been validated. This study aims to evaluate the reliability and validity of the GAD-7.Of 2,978 women who attended their first perinatal care visit and had the GAD-7 screening, 946 had a Composite International Diagnostic Interview (CIDI. The Cronbach's alpha was calculated to examine the reliability. We assessed the criterion validity by calculating operating characteristics. The construct validity was evaluated using factor analysis and association with health status on the CIDI. The cross-cultural validity was explored using the Rasch Rating Scale Model (RSM.The reliability of the GAD-7 was good (Cronbach's alpha = 0.89. A cutoff score of 7 or higher, maximizing the Youden Index, yielded a sensitivity of 73.3% and a specificity of 67.3%. One-factor structure of the GAD-7 was confirmed by exploratory and confirmatory factor analysis. Concurrent validity was supported by the evidence that higher GAD-7 scores were associated with poor self-rated physical and mental health. The Rasch RSM further confirmed the cross-cultural validity of the GAD-7.The results suggest that the Spanish-language version of the GAD-7 may be used as a screening tool for pregnant Peruvian women. The GAD-7 has good reliability, factorial validity, and concurrent validity. The optimal cutoff score obtained by maximizing the Youden Index should be considered cautiously; women who screened positive may require further investigation to confirm GAD diagnosis.

  6. A paleointensity test of the geocentric axial dipole (GAD) hypothesis (United States)

    Veikkolainen, Toni; Heimpel, Moritz; Evans, Michael E.; Pesonen, Lauri J.; Korhonen, Kimmo


    The geocentric axial dipole (GAD) model is central to many aspects of geophysics, including plate tectonics and paleoclimate. But its validity is by no means firmly established, particularly for the Precambrian. One test that has met with some success involves the distribution of paleomagnetic inclination angles. It works because any given field morphology has its own distinct probability distribution function (PDF) against which data compilations can be tested. Here, we investigate a second possible test using published paleointensity data. Once again, any given field morphology has a specific PDF of intensity. Likely field models consist of an underlying GAD on which is superimposed modest zonal quadrupole and octupole components. The corresponding paleointensity PDFs turn out to have more complicated shapes than their inclination counterparts, often having multiple maxima and minima. Given sufficient data, this complexity offers greater discrimination between models. In this paper, the potential of the paleointensity test is assessed using an extension of the PINT paleointensity database. We found it useful to analyse the Phanerozoic and Precambrian intervals separately. Despite the inherent limitations of this kind of analysis, a tripartite geodynamo with small zonal multipoles appears to be a good starting point on a way towards more fine-tuned models.

  7. No Contribution of GAD-65 and IA-2 Autoantibodies around Time of Diagnosis to the Increasing Incidence of Juvenile Type 1 Diabetes

    DEFF Research Database (Denmark)

    Thorsen, Steffen U.; Pipper, Christian B.; Mortensen, Henrik B.;


    Aims. A new perspective on autoantibodies as pivotal players in the pathogenesis of type 1 diabetes (T1D) has recently emerged. Our key objective was to examine whether increased levels of autoantibodies against the β-cell autoantigens glutamic acid decarboxylase (isoform 65) (GADA) and insulinom...

  8. Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies

    DEFF Research Database (Denmark)

    Pörksen, Sven; Laborie, Lene; Nielsen, Lotte


    BACKGROUND:To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (I...

  9. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin. (United States)

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki


    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.

  10. Biosynthetic arginine decarboxylase in phytopathogenic fungi. (United States)

    Khan, A J; Minocha, S C


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

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

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Ealick, Steven E. (Cornell)


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

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

    Energy Technology Data Exchange (ETDEWEB)

    Macrae, M.; Coffino, P. [Univ. of California, San Francisco, CA (United States); Plasterk, R.H.A. [Netherlands Cancer Institute, Amsterdam (Netherlands)


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

  13. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong


    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  14. Use of the Penn State Worry Questionnaire to Identify Individuals with Gad: An Indian Perspective

    Directory of Open Access Journals (Sweden)

    Parikh S


    Full Text Available 1.1.Background and Objectives: The Penn State Worry Questionnaire (PSWQ has been established as an efficacious tool to identify individuals with Generalized Anxiety Disorder (GAD as against those with other anxiety disorders. We report on the use of the PSWQ in the Indian population to identify individuals with GAD. 1.2.Methods: 30 individuals with GAD and 60 with other anxiety disorders without GAD completed the PSWQ. 1.3.Results: Using the Receiver Operating Characteristic curve analysis, a score of 64, which optimized both sensitivity and specificity, was found to discriminate between individuals with GAD and those with other anxiety disorders without GAD. 1.4.Limitations:The study conducted on a small sample would benefit from further research to establish the PSWQ as an efficacious tool for identify individuals with GAD. 1.5.Conclusion: Results from the current study offer strong support for the use of the PSWQ for initial screening and identification of individuals with GAD who present for treatment for anxiety disorders.

  15. 用ENDEAVOUR软件鉴定肥胖症致病基因GAD2%Verification of candidate gene GAD2 for human obesity by ENDEAVOUR

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 张小白


    Disease genes identification is very important for understanding disease pathogenesis and improving clinic treatment. The computational gene prioritization tools have the advantages of huge data sources, efficient data processing and low costs. Many researchers have studied the disputing candidate disease gene GAD2 for obesity on chromosome 1 Op 12 by association studies but obtained contradictory conclusions. In this paper, the probability of gene GAD2 being an obesity disease gene is verified by the computational gene prioritization tool ENDEAVOUR. The prioritization strategies of ENDEAVOUR are based on the similarity between the candidate gene and the profile derived from genes already known to be involved in the processes (the training genes) by genomic data fusion. In the computational results, the first rank of gene GAD2 means that the probability of gene GAD2 being an obesity disease gene is high. The results are helpful to explain the biological mechanism of gene GAD2 and genetic factors of the complex obesity disease..%鉴别复杂疾病致病基因对于治疗和预防疾病作用非常重要,致病基因预测软件是鉴别致病基因的有效工具.位于染色体10p12的基因GAD2是尚有争议的肥胖基因,许多研究者用实验方法研究基因GAD2,却得出了不一致的结论.基于生物计算的致病基因预测软件能弥补实验的不足,已成为揭示疾病发病机理和预测致病基因的有效途径,本文采用ENDEAVOUR软件来验证基因GAD2是否为肥胖症致病基因,该软件运用多种生物数据,根据每一个候选基因与已知致病基因的相似程度以排序,结果在35个候选基因中,基因GAD2排在最前面,表明基因GAD2最可能是肥胖症致病基因.预测结果有利于重新解释基因GAD2的生物功能,促使肥胖症药物的研制及治疗水平的提高.

  16. Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus (United States)

    Cabezas, Carolina; Irinopoulou, Theano; Cauli, Bruno; Poncer, Jean Christophe


    Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2–4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage. PMID:23565079

  17. Experimental Evidence and In Silico Identification of Tryptophan Decarboxylase in Citrus Genus

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    Luigi De Masi


    Full Text Available Plant tryptophan decarboxylase (TDC converts tryptophan into tryptamine, precursor of indolealkylamine alkaloids. The recent finding of tryptamine metabolites in Citrus plants leads to hypothesize the existence of TDC activity in this genus. Here, we report for the first time that, in Citrus x limon seedlings, deuterium labeled tryptophan is decarboxylated into tryptamine, from which successively deuterated N,N,N-trimethyltryptamine is formed. These results give an evidence of the occurrence of the TDC activity and the successive methylation pathway of the tryptamine produced from the tryptophan decarboxylation. In addition, with the aim to identify the genetic basis for the presence of TDC, we carried out a sequence similarity search for TDC in the Citrus genomes using as a probe the TDC sequence reported for the plant Catharanthus roseus. We analyzed the genomes of both Citrus clementina and Citrus sinensis, available in public database, and identified putative protein sequences of aromatic l-amino acid decarboxylase. Similarly, 42 aromatic l-amino acid decarboxylase sequences from 23 plant species were extracted from public databases. Potential sequence signatures for functional TDC were then identified. With this research, we propose for the first time a putative protein sequence for TDC in the genus Citrus.

  18. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii. (United States)

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia


    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  19. Characterization of the Entamoeba histolytica ornithine decarboxylase-like enzyme.

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    Anupam Jhingran

    Full Text Available BACKGROUND: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC, the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. METHODOLOGY AND RESULTS: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF of approximately 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size approximately 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO, an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. CONCLUSION: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from

  20. Ascending Midbrain Dopaminergic Axons Require Descending GAD65 Axon Fascicles for Normal Pathfinding

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    Claudia Marcela Garcia-Peña


    Full Text Available The Nigrostriatal pathway (NSP is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

  1. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA. (United States)

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D


    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  2. 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)


    The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

  3. Insomnia and generalized anxiety disorder: effects of cognitive behavior therapy for gad on insomnia symptoms. (United States)

    Bélanger, Lynda; Morin, Charles M; Langlois, Frédéric; Ladouceur, Robert


    Although clinical practice suggests that sleep complaints are frequent among patients with generalized anxiety disorder (GAD), frequency, severity, types of insomnia complaints, and relationship to GAD diagnosis severity in patients diagnosed using Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria are not well documented. Clinical data about the impact on insomnia symptoms of treating GAD worries are also lacking. The present study examined these aspects in 44 GAD patients who participated in a treatment study specifically addressing excessive worries through CBT interventions. All patients were assessed using a structured clinical interview and the Anxiety Disorder Interview Schedule-IV (ADIS-IV). They also completed anxiety and insomnia inventories, including the Insomnia Severity Index (ISI), a self-report measure which assesses insomnia type, severity and interference with daily life. Among this sample, 47.7% reported difficulties initiating sleep, 63.6% reported difficulties maintaining sleep, and 56.8% complained of waking too early in the morning. The majority of these patients (86.5%) reported never having experienced insomnia without having excessive worries. However, insomnia severity and GAD severity were not correlated. In this sample, patients with severe GAD did not necessarily report more severe insomnia symptoms. Regarding treatment impact on insomnia complaints, ISI post-treatment scores were significantly lower after treatment. Mean post-treatment scores almost reached ISI's "absence of clinical insomnia" category. Results indicate that this CBT package for GAD does have a significant impact on sleep quality even if sleep disturbances were not specifically addressed during treatment.

  4. Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

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


    Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae...... 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...

  5. Keto-isovalerate decarboxylase enzymes and methods of use thereof (United States)

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian


    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.

  6. Gamma-aminobutyric acid (GABA) and neuropeptides in neural areas mediating motion-induced emesis (United States)

    Damelio, F.; Daunton, Nancy G.; Fox, Robert A.


    Immunocytochemical methods were employed to localize the neurotransmitter amino acid gamma-aminobutyric acid and the neuropeptides substance P and Met-enkephalin in the area postrema (AP), area subpostrema (ASP), nucleus of the tractus solitarius (NTS), dorsal motor nucleus of the vagus nerve (DMNV), and lateral vestibular nucleus (LVN). Glutamic acid decarboxylase immunoreactive (GAD-IR) terminals and fibers were observed in the AP and particularly in the ASP. A gradual decrease in the density of terminals was seen towards the solitary complex. The DMNV revealed irregularly scattered GAD-IR terminals within the neuropil or closely surrounding neuronal cell bodies. The LVN, particularly the dorsal division, showed numerous axon terminals which were mostly localize around large neurons and their proximal dendrites. Substance P immunoreactive (SP-IR) terminals and fibers showed high density in the solitary complex, in particular within the lateral division. The ASP showed medium to low density of SP-IR fibers and terminals. The AP exhibited a small number of fibers and terminals irregularly distributed. The DMNV revealed a high density of SP-IR terminals and fibers that were mainly concentrated in the periphery. Very few terminals were detected in the LVN. Met-enkephalin immunoreactive (Met-Enk-IR) fibers and terminals showed high density and uniform distribution in the DMNV. Scattered terminals and fibers were observed in the AP, ASP, and NTS (particularly the lateral division). The very few fibers were observed in the LVN surrounded the neuronal cell bodies. The present report is part of a study designed to investigate the interaction between neuropeptides and conventional neurotransmitters under conditions producing motion sickness and in the process of sensory-motor adaptation.

  7. Characterization of a second ornithine decarboxylase isolated from Morganella morganii. (United States)

    De Las Rivas, Blanca; González, Ramón; Landete, José María; Muñoz, Rosario


    The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

  8. 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.

  9. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus 1 (United States)

    Legaz, María Estrella; Vicente, Carlos


    Arginase (EC, l-arginine decarboxylase (EC, and agmatine amidinohydrolase (EC activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID:16662821

  10. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus. (United States)

    Legaz, M E; Vicente, C


    Arginase (EC, l-arginine decarboxylase (EC, and agmatine amidinohydrolase (EC activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation.

  11. Homing of GAD65 specific autoimmunity and development of insulitis requires expression of both DQ8 and human GAD65 in transgenic mice (United States)

    Elagin, Raya B.; Balijepalli, Sadguna; Diacovo, Maria J.; Baekkeskov, Steinunn; Jaume, Juan C.


    MHC-class II genes determine susceptibility in human type-1 diabetes. In their context, presentation of target antigen(s) results in autoimmunity and β-cell destruction. An animal model, in which human β-cell autoantigen(s) are presented to effector-cells in the context of human MHC-class II diabetes susceptibility genes, would be desirable for studying molecular mechanisms of disease and developing antigen-specific immune-interventions. We report the development of antigen-specific insulitis in double-transgenic mice carrying the HLA-DQ8 diabetes susceptibility haplotype and expressing the human autoantigen GAD65 in pancreatic β-cells. Immunization with human GAD65 cDNA resulted in severe insulitis and low antibody levels in double-transgenic mice while control mice were mostly insulitis free. CFA/protein immunization resulted in high antibody levels and modest insulitis. Pancreatic lymphocytic infiltration progressed through stages (exocrine pancreas followed by peri and intra-insulitis). Adoptive transfer of splenocytes from DNA-immunized mice resulted in development of insulitis in recipient transgenics. Our results show that immunization with a clinically relevant, type-1 diabetes human autoantigen, in a humanized genetic setting, results in the development of an immune response that homes to islets of Langerhans. This animal model will facilitate studies of autoimmunity to GAD65 in the context of HLA-DQ8, and development of methods to induce tolerance and prevent insulitis. PMID:19289270

  12. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L. (United States)

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D


    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  13. Role of ornithine decarboxylase in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Wensheng Deng; Xian Jiang; Yu Mei; Jingzhong Sun; Rong Ma; Xianxi Liu; Hui Sun; Hui Tian; Xueying Sun


    Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis that decarboxylates ornithine to putrescine, has become a promising target for cancer research. The aim of this study is to investigate the role of ODC in breast cancer. We detected expression of ODC in breast cancer tissues and four breast cancer cell lines, and transfected breast cancer cells with an adenoviral vector carrying antisense ODC (rAd-ODC/Ex3as) and examined their growth and migration.ODC was overexpressed in breast cancer tissues and cell lines compared with non-tumor tissues and normal breast epithelial celis,and there was a positive correlation between the level of ODC mRNA and the staging of tumors.The expression of ODC correlated with cyclin D1,a cell cycle protein,in synchronized breast cancer MDA-MB-231 cells.Gene transfection of rAd-ODC/Ex3as markedly down-regulated expression Of ODC and cyclin D1,resulting in suppression of proliferation and cell cycle arrest at G0-G1 phase,and the inhibifion of colony formation,an anchorage-independent growth pattern,and the migratory ability of MDA-MB-231 cells.rAd-ODC/Ex3as also markedly reduced the concentration of putrescine,but not spermidine or spermine,in MDA-MB-231 cells.The results suggested that the ODC gene might act as aprognostic factor for breast cancer and it could be a promising therapeutic target.

  14. Benzodiazepine Discontinuation among Adults with GAD: A Randomized Trial of Cognitive-Behavioral Therapy (United States)

    Gosselin, Patrick; Ladouceur, Robert; Morin, Charles M.; Dugas, Michel J.; Baillargeon, Lucie


    This study evaluated the specific effectiveness of cognitive-behavior therapy (CBT) combined with medication tapering for benzodiazepine discontinuation among generalized anxiety disorder (GAD) patients by using a nonspecific therapy control group. Sixty-one patients who had used benzodiazepines for more than 12 months were randomly assigned to…

  15. Diasporicità sull’esempio di Erich Auerbach in Gad Lerner e Miro Silvera

    NARCIS (Netherlands)

    Jansen, M.M.; Arts, Clemens


    A partire dall’esilio di Erich Auerbach a Istanbul dal 1936 al 1947, il contributo propone una riflessione sui concetti di diaspora e di esilio da adattare a due scrittori italo‐ebrei provenienti dalla diaspora orientale: Miro Silvera con Il passeggero occidentale (2009) e Gad Lerner con Scintille (

  16. The rice R2R3-MYB transcription factor OsMYB55 is involved in the tolerance to high temperature and modulates amino acid metabolism.

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    Ashraf El-Kereamy

    Full Text Available Temperatures higher than the optimum negatively affects plant growth and development. Tolerance to high temperature is a complex process that involves several pathways. Understanding this process, especially in crops such as rice, is essential to prepare for predicted climate changes due to global warming. Here, we show that OsMYB55 is induced by high temperature and overexpression of OsMYB55 resulted in improved plant growth under high temperature and decreased the negative effect of high temperature on grain yield. Transcriptome analysis revealed an increase in expression of several genes involved in amino acids metabolism. We demonstrate that OsMYB55 binds to the promoter regions of target genes and directly activates expression of some of those genes including glutamine synthetase (OsGS1;2 glutamine amidotransferase (GAT1 and glutamate decarboxylase 3 (GAD3. OsMYB55 overexpression resulted in an increase in total amino acid content and of the individual amino acids produced by the activation of the above mentioned genes and known for their roles in stress tolerance, namely L-glutamic acid, GABA and arginine especially under high temperature condition. In conclusion, overexpression of OsMYB55 improves rice plant tolerance to high temperature, and this high tolerance is associated with enhanced amino acid metabolism through transcription activation.

  17. Conformational stabilization of rat s-adenosylmethionine decarboxylase by putrescine. (United States)

    Wada, Makiko; Shirahata, Akira


    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC α-subunit, while there was no incorporation of IAA in the β-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of α-subunit and 0.9 mol of IAA/mol of β-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

  18. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots. (United States)

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


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

  19. 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


    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.

  20. Midkine, heparin-binding growth factor, blocks kainic acid-induced seizure and neuronal cell death in mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Lim In J


    Full Text Available Abstract Background Midkine (MK, a member of the heparin-binding growth factor family, which includes MK and pleiotrophin, is known to possess neurotrophic and neuroprotective properties in the central nervous system. Previous studies have shown that MK is an effective neuroprotective agent in reducing retinal degeneration caused by excessive light and decreasing hippocampal neuronal death in ischemic gerbil brain. The present study was undertaken to investigate whether MK acts as an anticonvulsant in kainic acid (KA-induced seizure in mouse and blocks KA-mediated neuronal cell death in hippocampus. Results Increased expression of MK was found in hippocampus of mouse following seizures induced by intracerebroventricular injection of KA, and MK expression was found in glial fibrillary acidic protein (GFAP-positive astrocytes. Concurrent injection of MK and KA attenuated KA-induced seizure activity and cell death of hippocampal neurons including pyramidal cells and glutamic acid decarboxylase 67 (GAD67-positive GABAergic interneurons in the CA3 and hilar area. Conclusion The results of the present study indicate that MK functions as an anticonvulsant and neuroprotective agent in hippocampus during KA-induced seizures.

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

    DEFF Research Database (Denmark)

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga


    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...

  2. Genetics Home Reference: aromatic l-amino acid decarboxylase deficiency (United States)

    ... the limbs (athetosis). They may be lacking in energy (lethargic), feed poorly, startle easily, and have sleep disturbances. People with AADC deficiency may also experience episodes called oculogyric crises that involve abnormal rotation of the eyeballs; extreme ...

  3. Dietary supplementation with n-3 fatty acids from weaning limits brain biochemistry and behavioural changes elicited by prenatal exposure to maternal inflammation in the mouse model (United States)

    Li, Q; Leung, Y O; Zhou, I; Ho, L C; Kong, W; Basil, P; Wei, R; Lam, S; Zhang, X; Law, A C K; Chua, S E; Sham, P C; Wu, E X; McAlonan, G M


    Prenatal exposure to maternal immune activation (MIA) increases the risk of schizophrenia and autism in the offspring. The MIA rodent model provides a valuable tool to directly test the postnatal consequences of exposure to an early inflammatory insult; and examine novel preventative strategies. Here we tested the hypotheses that behavioural differences in the MIA mouse model are accompanied by in vivo and ex vivo alterations in brain biochemistry; and that these can be prevented by a post-weaning diet enriched with n-3 polyunsaturated fatty acid (PUFA). The viral analogue PolyI:C (POL) or saline (SAL) was administered to pregnant mice on gestation day 9. Half the resulting male offspring (POL=21; SAL=17) were weaned onto a conventional lab diet (n-6 PUFA); half were weaned onto n-3 PUFA-enriched diet. In vivo magnetic resonance spectroscopy measures were acquired prior to behavioural tests; glutamic acid decarboxylase 67 (GAD67) and tyrosine hydroxylase protein levels were measured ex vivo. The main findings were: (i) Adult MIA-exposed mice fed a standard diet had greater N-acetylaspartate/creatine (Cr) and lower myo-inositol/Cr levels in the cingulate cortex in vivo. (ii) The extent of these metabolite differences was correlated with impairment in prepulse inhibition. (iii) MIA-exposed mice on the control diet also had higher levels of anxiety and altered levels of GAD67 ex vivo. (iv) An n-3 PUFA diet prevented all the in vivo and ex vivo effects of MIA observed. Thus, n-3 PUFA dietary enrichment from early life may offer a relatively safe and non-toxic approach to limit the otherwise persistent behavioural and biochemical consequences of prenatal exposure to inflammation. This result may have translational importance. PMID:26393487

  4. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes. (United States)

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho


    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  5. Defense of GAD during the 1950s and early 1960s (United States)

    Frankel, H. R.


    Paleomagnetists favoring continental offered empirical and theoretical support for the GAD hypothesis. Initial support came from the discovery that the mean directions of rock units, regardless of polarity, laid down back through the Upper Tertiary centered on the rotational pole. Armed with Fisher's statistics, Hospers (1951, 1953) found that the mean direction of the NRM of Icelandic lava flows back through the Miocene better agreed with the GAD field than with the present field. Similarly, Campbell and Runcorn (1956), Creer (1956), and Irving and Green (1957) respectively found that the natural remanent magnetization of Late Tertiary Columbia River basalts, Quaternary basalts of Argentina, and Late Cenozoic New Volcanics of Victoria supported the hypothesis. If significant continental drift or "true" polar wander has occurred, paleomagnetic data alone cannot determine if the axial element of the GAD hypothesis holds earlier than Late Tertiary. Extending the GAD hypothesis back in time requires an approach involving a means independent of paleomagnetism for determining past latitudes. Irving was the first to realize that the paleoclimatology would work. If the GAD hypothesis holds, then paleolatitudes based on paleomagnetism and paleoclimatology should agree. Irving (1956) found that, except for the Squantum Tillite, the paleomagnetically and paleoclimatically determined paleolatitudes for Europe, North America, India, and Tasmania were in agreement. He concluded that the magnetic and rotational axes have coincided since the Paleozoic. Blackett (1961) also compared paleoclimatic and paleomagnetic data-sets. Irving and Briden (1962, 1964) further appealed to paleoclimatology to defend the hypothesis. Determining the paleolatitude spectra for several paleoclimatic indicators, they found the present latitude of fossil instances inconsistent with the latitude of modern instances while their paleomagnetically determined paleolatitudes, which assumed the GAD hypothesis

  6. GAD65 haplodeficiency conveys resilience in animal models of stress-induced psychopathology

    Directory of Open Access Journals (Sweden)

    Iris eMüller


    Full Text Available GABAergic mechanisms are critically involved in the control of fear and anxiety, but their role in the development of stress-induced psychopathologies, including post-traumatic stress disorder (PTSD and mood disorders is not sufficiently understood. We studied these functions in two established mouse models of risk factors for stress-induced psychopathologies employing variable juvenile stress and/or social isolation. A battery of emotional tests in adulthood revealed the induction of contextually generalized fear, anxiety, hyperarousal and depression-like symptoms in these paradigms. These reflect the multitude and complexity of stress effects in human PTSD patients. With factor analysis we were able to identify parameters that reflect these different behavioral domains in stressed animals and thus provide a basis for an integrated scoring of affectedness more closely resembling the clinical situation than isolated parameters. To test the applicability of these models to genetic approaches we further tested the role of GABA using heterozygous mice with targeted mutation of the GABA synthesizing enzyme GAD65 (GAD65+/- mice, which show a delayed postnatal increase in tissue GABA content in limbic and cortical brain areas. Unexpectedly, GAD65(+/- mice did not show changes in exploratory activity regardless of the stressor type and were after the variable juvenile stress procedure protected from the development of contextual generalization in an auditory fear conditioning experiment. Our data demonstrate the complex nature of behavioral alterations in rodent models of stress-related psychopathologies and suggest that GAD65 haplodeficiency, likely through its effect on the postnatal maturation of GABAergic transmission, conveys resilience to some of these stress-induced effects.

  7. Aberrant accumulation of the diabetes autoantigen GAD65 in Golgi membranes in conditions of ER stress and autoimmunity

    DEFF Research Database (Denmark)

    Phelps, Edward A; Cianciaruso, Chiara; Michael, Iacovos P


    , an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes....... The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen presenting cells and T cell stimulation compared to the non-palmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human beta cells in pancreatic sections from GAD65 autoantibody positive...... individuals, who have not yet progressed to clinical onset of T1D, and T1D patients with residual beta cell mass and ongoing T cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system following...

  8. Arginine decarboxylase as the source of putrescine for tobacco alkaloids (United States)

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


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

  9. Lotus hairy roots expressing inducible arginine decarboxylase activity. (United States)

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H


    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  10. COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Fenalti, Gustavo; Hampe, Christiane S.; Arafat, Yasir; Law, Ruby H.P.; Banga, J. Paul; Mackay, Ian R.; Whisstock, James C.; Buckle, Ashley M.; Rowley, Merrill J. (UWASH); (King’s College); (Monash)


    To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow 'molecular positioning' of epitope sites for B- and T-cell reactivity. Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate- and NH{sub 2}-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67. Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity.

  11. Attenuation of γ-aminobutyric acid (GABA) transaminase activity contributes to GABA increase in the cerebral cortex of mice exposed to β-cypermethrin. (United States)

    Han, Y; Cao, D; Li, X; Zhang, R; Yu, F; Ren, Y; An, L


    The current study investigated the γ-aminobutyric acid (GABA) levels and GABA metabolic enzymes (GABA transaminase (GABA(T)) and glutamate decarboxylase (GAD)) activities at 2 and 4 h after treatment, using a high-performance liquid chromatography with ultraviolet detectors and colorimetric assay, in the cerebral cortex of mice treated with 20, 40 or 80 mg/kg β-cypermethrin by a single oral gavage, with corn oil as vehicle control. In addition, GABA protein (4 h after treatment), GABA(T) protein (2 h after treatment) and GABA receptors messenger RNA (mRNA) expression were detected by immunohistochemistry, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction, respectively. β-Cypermethrin (80 mg/kg) significantly increased GABA levels in the cerebral cortex of mice, at both 2 and 4 h after treatment, compared with the control. Also, GABA immunohistochemistry results suggested that the number of positive granules was increased in the cerebral cortex of mice 4 h after exposure to 80 mg/kg β-cypermethrin when compared with the control. Furthermore, the results also showed that GABA(T) activity detected was significantly decreased in the cerebral cortex of mice 2 h after β-cypermethrin administration (40 or 80 mg/kg). No significant changes were found in GAD activity, or the expression of GABA(T) protein and GABAB receptors mRNA, in the cerebral cortex of mice, except that 80 mg/kg β-cypermethrin caused a significant decrease, compared with the vehicle control, in GABAA receptors mRNA expression 4 h after administration. These results suggested that attenuated GABA(T) activity induced by β-cypermethrin contributed to increased GABA levels in the mouse brain. The downregulated GABAA receptors mRNA expression is most likely a downstream event.

  12. Current theoretical models of generalized anxiety disorder (GAD): conceptual review and treatment implications. (United States)

    Behar, Evelyn; DiMarco, Ilyse Dobrow; Hekler, Eric B; Mohlman, Jan; Staples, Alison M


    Theoretical conceptualizations of generalized anxiety disorder (GAD) continue to undergo scrutiny and refinement. The current paper critiques five contemporary models of GAD: the Avoidance Model of Worry and GAD [Borkovec, T. D. (1994). The nature, functions, and origins of worry. In: G. Davey & F. Tallis (Eds.), Worrying: perspectives on theory assessment and treatment (pp. 5-33). Sussex, England: Wiley & Sons; Borkovec, T. D., Alcaine, O. M., & Behar, E. (2004). Avoidance theory of worry and generalized anxiety disorder. In: R. Heimberg, C. Turk, & D. Mennin (Eds.), Generalized anxiety disorder: advances in research and practice (pp. 77-108). New York, NY, US: Guilford Press]; the Intolerance of Uncertainty Model [Dugas, M. J., Letarte, H., Rheaume, J., Freeston, M. H., & Ladouceur, R. (1995). Worry and problem solving: evidence of a specific relationship. Cognitive Therapy and Research, 19, 109-120; Freeston, M. H., Rheaume, J., Letarte, H., Dugas, M. J., & Ladouceur, R. (1994). Why do people worry? Personality and Individual Differences, 17, 791-802]; the Metacognitive Model [Wells, A. (1995). Meta-cognition and worry: a cognitive model of generalized anxiety disorder. Behavioural and Cognitive Psychotherapy, 23, 301-320]; the Emotion Dysregulation Model [Mennin, D. S., Heimberg, R. G., Turk, C. L., & Fresco, D. M. (2002). Applying an emotion regulation framework to integrative approaches to generalized anxiety disorder. Clinical Psychology: Science and Practice, 9, 85-90]; and the Acceptance-based Model of GAD [Roemer, L., & Orsillo, S. M. (2002). Expanding our conceptualization of and treatment for generalized anxiety disorder: integrating mindfulness/acceptance-based approaches with existing cognitive behavioral models. Clinical Psychology: Science and Practice, 9, 54-68]. Evidence in support of each model is critically reviewed, and each model's corresponding evidence-based therapeutic interventions are discussed. Generally speaking, the models share an

  13. Comparative Autonomic Responses to Diagnostic Interviewing between Individuals with GAD, MDD, SAD and Healthy Controls (United States)

    Diamond, Allison E.; Fisher, Aaron J.


    Dysregulation of the autonomic nervous system (ANS) has been well documented in individuals diagnosed with a range of psychological disorders, including generalized anxiety disorder (GAD) and major depressive disorder (MDD). Moreover, these disorders both confer an increased risk of cardiovascular disease—which may relate to increased sympathetic and decreased parasympathetic tone. Extant research has indicated a reduction in autonomic flexibility in GAD, and while reduced flexibility has also been seen in MDD, the specific physiological alterations have been more difficult to categorize due to methodological limitations, including high co-morbidity rates with anxiety disorders. Prior studies have largely assessed autonomic functioning in stress paradigms or at the trait level, yet to date, no research has investigated the ANS during a diagnostic interview, a ubiquitous task employed in both research and clinical settings. In this study we sought to identify physiological differences in both branches of the ANS across diagnostic categories in the context of a diagnostic interview. Participants (n = 82) were administered a structured clinical interview, during which heart rate (HR), respiratory sinus arrhythmia (RSA) and pre-ejection period (PEP) were recorded in participants carrying a diagnosis of GAD (n = 34), MDD (n = 22), Social Anxiety Disorder (SAD; n = 15) and healthy controls (n = 27). Person-specific linear regression models were employed to assess the level and slope for HR, RSA and PEP throughout the course of the interview. A multivariate analysis of variance (MANOVA) model was conducted to baseline differences in HR, RSA and PEP between diagnostic groups. Multiple regression models were then conducted to differences in slope of HR, RSA and PEP throughout the course of the interview amongst diagnostic groups, including both suppression and worry as moderators. Results indicated significant increases in RSA throughout the interview in MDD (p = 0

  14. GABA and its B-receptor are present at the node of Ranvier in a small population of sensory fibers, implicating a role in myelination

    DEFF Research Database (Denmark)

    Corell, Mikael; Wicher, Grzegorz; Radomska, Katarzyna J;


    of the GABAB receptor, GABA, and glutamic acid decarboxylase GAD65/67 in both development and injury in fetal dissociated dorsal root ganglia (DRG) cell cultures and in the rat sciatic nerve. We found that GABA, GAD65/67, and the GABAB receptor were expressed in premyelinating and nonmyelinating Schwann cells...

  15. Acid Tolerance Response of Anaerobic Sludge with Butyric Acid Stress during the Enhanced Biohydrogen Process%生物产氢过程中厌氧污泥耐酸响应的生物化学机制

    Institute of Scientific and Technical Information of China (English)

    孟影; 张光生; 王爱杰; 严群


    During the anaerobic digestion of biomass,large amounts of organic acids were produced. This results in an inhibitory effect on anaerobic microbiology,which in turn inhibits large-scale production of hydrogen. In this study, butyric acid was use as stress on the sludge, and it was found that the production rates of butyric acid, acetic acid and hydrogenwas reached at 400 mmol/mol, 1100 mmol/mol, 3690 mL/mol, respectively, which was higher 110%, 54% and 65% than that of the corresponding values in the control group. Moreover, glutamate acid decarboxylase (GAD) activity, dehydrogenase activity and the content of DNA was in-creased to 11. 6 ,μm/(g · TS · h), 6982. 12 μg TF/(g · TS · h) 14. 72 ng/mL, with an increment of 48%, 50%, 10.7%, respectively, and the content of extracellular polymeric substances (EPS) have improved significantly, loose bound protein, loose bound polysaccharide, tight bound protein and tight bound polysaccharide content were 147%, 34. 8%, 35%, 21. 6% higher than that of the control. The results demonstrate that the appropriate concentration of butyric acid stress on sludge can excite acid tolerance response (Acid tolerance response, ATR) and to improve acid tolerance of sludge, and improve the efficiency of anaerobic hydrogen production.%生物质厌氧发酵产氢过程中积累的大量酸性物质,会对厌氧微生物产生抑制作用,进而制约氢气的持续产生.作者采用不同浓度丁酸对污泥进行胁迫,结果表明:当丁酸胁迫质量浓度为6 g/L时,污泥厌氧发酵过程中丁酸、乙酸以及氢气产量最高,分别达到1 071 mmol/mol,462mmol/mol和3 690 mL/mol,比对照组分别提高了110%,54%和65%;此外,产氢过程中谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)、脱氢酶以及DNA总量活性最高达到11.6μmol/(g·TS·h),6 982.12 μg TF/(g·TS·h),14.72 ng/mL,相对于对照组分别提高了48%,50%,10.7%;同时,经过酸胁迫后,

  16. Cognitive Control and Anxiety Disorders: Metacognitive Beliefs and Strategies of Control Thought in GAD and OCD

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Pérez Nieto


    Full Text Available En el presente trabajo se asume la relevancia que los procesos de control cognitivo pueden tener en trastornos de ansiedad como el GAD o el TOC. Se pretende identificar las creencias metacognitivas derivadas del modelo S-REF (Wells y Mathews, 1996, Wells, 2000 que se vincularán en mayor medida al espectro del trastorno de ansiedad generalizada y del trastorno obsesivo-compulsivo y el efecto que dichas creencias pueden tener en el uso de estrategias de control cognitivo. Para ello, una muestra de 75 participantes, 24 con diagnósticod de GAD o de TOC, y 51 sin diagnóstico mentales, fueron evaluados mediante el MCQ-30 y el TCQ. El ANOVA entre los grupos permitió encontrar significativas las mayores puntuaciones en creencias sobre la peligrosidad de no controlar las preocupaciones por parte de los participantes con trastorno de ansiedad. Los análisis de regresión permitieron comprobar que ese tipo de creencias favorecían, además, el uso de estrategias de control cognitivo poco adaptativas, como el castigo.

  17. Using the GAD-Q-IV to identify generalized anxiety disorder in psychiatric treatment seeking and primary care medical samples. (United States)

    Moore, Michael T; Anderson, Nicholas L; Barnes, Jill M; Haigh, Emily A P; Fresco, David M


    The fourth edition of the Generalized Anxiety Disorder Questionnaire (GAD-Q-IV) is a self-report measure that is commonly used to screen for the presence of generalized anxiety disorder (GAD). The current investigation attempted to identify an optimal cut score using samples obtained from an outpatient psychiatric (n=163) and primary care clinic (n=99). Results indicated that a cut score of 7.67 provided an optimal balance of sensitivity (.85) and specificity (.74) comparable to a previously identified cut score (5.7) across both samples (sensitivity=.90, specificity=.66). However, both cut scores were consistently outperformed by a score representing the criteria for GAD described in the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (sensitivity=.89, specificity=.82).

  18. Cell biology, physiology and enzymology of phosphatidylserine decarboxylase. (United States)

    Di Bartolomeo, Francesca; Wagner, Ariane; Daum, Günther


    Phosphatidylethanolamine is one of the most abundant phospholipids whose major amounts are formed by phosphatidylserine decarboxylases (PSD). Here we provide a comprehensive description of different types of PSDs in the different kingdoms of life. In eukaryotes, type I PSDs are mitochondrial enzymes, whereas other PSDs are localized to other cellular compartments. We describe the role of mitochondrial Psd1 proteins, their function, enzymology, biogenesis, assembly into mitochondria and their contribution to phospholipid homeostasis in much detail. We also discuss briefly the cellular physiology and the enzymology of Psd2. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

  19. Pantothenic acid biosynthesis in zymomonas

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.


    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  20. 米糠中γ-氨基丁酸的富集及纯化工艺研究%Study on enrichment and purification of γ-aminobutyric acid in rice bran

    Institute of Scientific and Technical Information of China (English)

    管娜娜; 张晖; 王立; 郭晓娜; 钱海峰


    The accumulation of GABA in rice bran by glutamate decarboxylase(GAD) ,and the purification of GABA enriched solution using cation ion exchange resin were investigated. The optimal accumulation conditions of GABA were determined as described below: the ionic strength of citric acid-disodium hydrogen phosphate buffer solution was 0.02mol/L, the reaction time was 16h. Under the optimal conditions, the concentration of GABA reached 2900mg/100g rice bran. The optimal purification conditions of GABA enriched solution also be determined as following: the optimal pH of sample solution was 2.0, the concentration of GABA enriched solution was 2.0mg/mL,concentration of eluting agent was 2.0mol/L. And the final purity of GABA reached 61.25%.%以米糠为原料,利用米糠中高活性谷氨酸脱羧酶(GAD)进行γ-氨基丁酸(GABA)的富集实验,并采用阳离子交换树脂对富集液中GABA进行分离纯化.结果表明:采用0.02mol/L Ph5.6的Na2HPO4-柠檬酸缓冲液进行GABA富集实验,反应16h后可得到GABA 2900mg/100g米糠.采用D001大孔强酸性阳离子交换树脂对该富集液进行纯化实验,调节富集液Ph2.0,以2mg/Ml的浓度上样吸附,2mol/L的氨水浓度进行洗脱,最终可得γ-氨基丁酸纯度61.25%.

  1. Cultural adaptation into Spanish of the generalized anxiety disorder-7 (GAD-7 scale as a screening tool

    Directory of Open Access Journals (Sweden)

    Pérez-Páramo María


    Full Text Available Abstract Background Generalized anxiety disorder (GAD is a prevalent mental health condition which is underestimated worldwide. This study carried out the cultural adaptation into Spanish of the 7-item self-administered GAD-7 scale, which is used to identify probable patients with GAD. Methods The adaptation was performed by an expert panel using a conceptual equivalence process, including forward and backward translations in duplicate. Content validity was assessed by interrater agreement. Criteria validity was explored using ROC curve analysis, and sensitivity, specificity, predictive positive value and negative value for different cut-off values were determined. Concurrent validity was also explored using the HAM-A, HADS, and WHO-DAS-II scales. Results The study sample consisted of 212 subjects (106 patients with GAD with a mean age of 50.38 years (SD = 16.76. Average completion time was 2'30''. No items of the scale were left blank. Floor and ceiling effects were negligible. No patients with GAD had to be assisted to fill in the questionnaire. The scale was shown to be one-dimensional through factor analysis (explained variance = 72%. A cut-off point of 10 showed adequate values of sensitivity (86.8% and specificity (93.4%, with AUC being statistically significant [AUC = 0.957-0.985; p 0.001. Limitations Elderly people, particularly those very old, may need some help to complete the scale. Conclusion After the cultural adaptation process, a Spanish version of the GAD-7 scale was obtained. The validity of its content and the relevance and adequacy of items in the Spanish cultural context were confirmed.

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

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


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

  3. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity. (United States)

    Lietzan, Adam D; St Maurice, Martin


    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  4. Lack of Support for the Association Between GAD2 Polymorphisms andSevere Human Obesity

    Energy Technology Data Exchange (ETDEWEB)

    Swarbrick, Michael M.; Waldenmaier, Bjorn; Pennacchio, Len A.; Lind,Denise L.; Cavazos, Martha M.; Geller, Frank; Merriman, Raphael; Ustaszewska, Anna; Malloy, Mary; Scherag, Andre; Hsueh, Wen-Chi; Rief,Winfried; Mauvais-Jarvis, Franck; Pullinger, Clive R.; Kane, John P.; Dent, Robert; McPherson, Ruth; Kwok, Pui-Yan; Hinney, Anke; Hebebrand,Johannes; Vaisse, Christian


    Demonstration of association between common genetic variants and chronic human diseases such as obesity could have profound implications for the prediction, prevention and treatment of these conditions. Unequivocal proof of such an association, however, requires adherence to established methodological guidelines, which include independent replication of initial positive findings. Recently, single nucleotide polymorphisms (SNPs) within GAD2 were found to be associated with class III obesity (BMI > 40 kg/m2) in 188 families (612 individuals) segregating the condition and a case-control study of 575 cases and 646 lean controls. Functional data supporting a pathophysiological role for one of the SNPs (-243A>G) were also presented. In the present study, we attempted to replicate this association in larger groups of subjects, and to extend the functional studies of the -243A>G SNP. In 2,327 subjects comprising 692 German nuclear families with severe, early-onset obesity, we found no evidence for a relationship between the three GAD2 SNPs and obesity, whether SNPs were studied individually or as haplotypes. In two independent case-control studies (a total of 680 class III obesity cases and 1,186 lean controls), there was no significant relationship between the -243A>G SNP and obesity (odds ratio (OR) = 0.99, 95% CI 0.83 - 1.18,in the pooled sample). These negative findings were reinforced by a meta-analysis for the association between the 243G allele and class III obesity, which yielded an OR of 1.11 (95% CI 0.90 - 1.36) in a total sample of 1,252 class III obese cases and 1,800 lean controls. Finally,we were unable to confirm or extend the functional data pertaining to the -243A>G variant. Potential confounding variables in association studies involving common variants and complex diseases (low power to detect modest genetic effects, over-interpretation of marginal data, population stratification and biological plausibility) are also discussed in the context of GAD2 and

  5. γ-Aminobutyric Acid Imparts Partial Protection from Salt Stress Injury to Maize Seedlings by Improving Photosynthesis and Upregulating Osmoprotectants and Antioxidants (United States)

    Wang, Yongchao; Gu, Wanrong; Meng, Yao; Xie, Tenglong; Li, Lijie; Li, Jing; Wei, Shi


    γ-Aminobutyric acid (GABA) has high physiological activity in plant stress physiology. This study showed that the application of exogenous GABA by root drenching to moderately (MS, 150 mM salt concentration) and severely salt-stressed (SS, 300 mM salt concentration) plants significantly increased endogenous GABA concentration and improved maize seedling growth but decreased glutamate decarboxylase (GAD) activity compared with non-treated ones. Exogenous GABA alleviated damage to membranes, increased in proline and soluble sugar content in leaves, and reduced water loss. After the application of GABA, maize seedling leaves suffered less oxidative damage in terms of superoxide anion (O2·−) and malondialdehyde (MDA) content. GABA-treated MS and SS maize seedlings showed increased enzymatic antioxidant activity compared with that of untreated controls, and GABA-treated MS maize seedlings had a greater increase in enzymatic antioxidant activity than SS maize seedlings. Salt stress severely damaged cell function and inhibited photosynthesis, especially in SS maize seedlings. Exogenous GABA application could reduce the accumulation of harmful substances, help maintain cell morphology, and improve the function of cells during salt stress. These effects could reduce the damage to the photosynthetic system from salt stress and improve photosynthesis and chlorophyll fluorescence parameters. GABA enhanced the salt tolerance of maize seedlings. PMID:28272438

  6. A unique combination of autoimmune limbic encephalitis, type 1 diabetes, and Stiff person syndrome associated with GAD-65 antibody. (United States)

    Sharma, Chandra Mohan; Pandey, Rajendra Kumar; Kumawat, Banshi Lal; Khandelwal, Dinesh; Gandhi, Pankaj


    Antibodies to GAD-65 have been implicated in the pathogenesis of type 1 diabetes, limbic encephalitis and Stiff person syndrome, however these diseases rarely occur concurrently. We intend to present a rare case of 35 year old female who was recently diagnosed as having type 1 diabetes presented with 1½ month history of recurrent seizures, subacute onset gait ataxia, dysathria, psychiatric disturbance and cognitive decline. No tumor was found on imaging and the classic paraneoplastic panel was negative. Cerebrospinal fluid and blood was positive for GAD-65 antibodies. Patient showed significant improvement with immunomodulatory therapy. Association of GAD-65 antibodies has been found with various disorders including type 1 diabetes, limbic encephalitis, Stiff person syndrome, cerebellar ataxia and palatal myoclonus. This case presents with unique combination of type 1 diabetes, Stiff person syndrome and limbic encephalitis associated with GAD-65 antibodies that is responsive to immunotherapy. It also highlights the emerging concept of autoimmunity in the causation of various disorders and there associations.

  7. Characterization of the activity and expression of arginine decarboxylase in human and animal Chlamydia pathogens. (United States)

    Bliven, Kimberly A; Fisher, Derek J; Maurelli, Anthony T


    Chlamydia pneumoniae encodes a functional arginine decarboxylase (ArgDC), AaxB, that activates upon self-cleavage and converts l-arginine to agmatine. In contrast, most Chlamydia trachomatis serovars carry a missense or nonsense mutation in aaxB abrogating activity. The G115R missense mutation was not predicted to impact AaxB functionality, making it unclear whether AaxB variations in other Chlamydia species also result in enzyme inactivation. To address the impact of gene polymorphism on functionality, we investigated the activity and production of the Chlamydia AaxB variants. Because ArgDC plays a critical role in the Escherichia coli acid stress response, we studied the ability of these Chlamydia variants to complement an E. coli ArgDC mutant in an acid shock assay. Active AaxB was detected in four additional species: Chlamydia caviae, Chlamydia pecorum, Chlamydia psittaci, and Chlamydia muridarum. Of the C. trachomatis serovars, only E appears to encode active enzyme. To determine when functional enzyme is present during the chlamydial developmental cycle, we utilized an anti-AaxB antibody to detect both uncleaved and cleaved enzyme throughout infection. Uncleaved enzyme production peaked around 20 h postinfection, with optimal cleavage around 44 h. While the role ArgDC plays in Chlamydia survival or virulence is unclear, our data suggest a niche-specific function.

  8. Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16. (United States)

    Malvagia, S; Papi, L; Morrone, A; Donati, M A; Ciani, F; Pasquini, E; la Marca, G; Scholte, H R; Genuardi, M; Zammarchi, E


    Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

  9. Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

    NARCIS (Netherlands)

    Le Jeune, C.; Lonvaud-Funel, A.; Brink, B. ten; Hofstra, H.; Vossen, J.M.B.M. van der


    On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the h

  10. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. (United States)

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B


    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  11. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai


    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  12. Enzyme Architecture: The Activating Oxydianion Binding Domain for Orotidine 5′-Monophophate Decarboxylase (United States)

    Spong, Krisztina; Amyes, Tina L.; Richard, John P.


    Orotidine 5′-monophosphate decarboxylase catalyzes the decarboxylation of truncated substrate (1-β-D-erythrofuranosyl)orotic acid (EO) to form (1-β-D-erythrofuranosyl)uracil (EU). This enzymecatalyzed reaction is activated by tetrahedral oxydianions, which bind weakly to unliganded OMPDC and tightly to the enzyme-transition state complex, with the following intrinsic oxydianion binding energies (kcal/mole): SO32−, −8.3; HPO32−, −7.7; S2O32−, −4.6; SO42−, −4.5; HOPO32−, −3.0; HOAsO32−, no activation detected. We propose that oxydianion and orotate binding domains perform complementary functions in catalysis of decarboxylation reactions. (1) The orotate binding domain carries out decarboxylation of the orotate ring. (2) The activating oxydianion binding domain has the cryptic function of utilizing binding interactions with tetrahedral inorganic oxydianions to drive an enzyme conformational change that results in the stabilization of transition states at the distant orotate domain. PMID:24274746

  13. Enzyme architecture: the activating oxydianion binding domain for orotidine 5'-monophophate decarboxylase. (United States)

    Spong, Krisztina; Amyes, Tina L; Richard, John P


    Orotidine 5'-monophosphate decarboxylase catalyzes the decarboxylation of truncated substrate (1-β-D-erythrofuranosyl)orotic acid to form (1-β-D-erythrofuranosyl)uracil. This enzyme-catalyzed reaction is activated by tetrahedral oxydianions, which bind weakly to unliganded OMPDC and tightly to the enzyme-transition state complex, with the following intrinsic oxydianion binding energies (kcal/mol): SO3(2-), -8.3; HPO3(2-), -7.7; S2O3(2-), -4.6; SO4(2-), -4.5; HOPO3(2-), -3.0; HOAsO3(2-), no activation detected. We propose that the oxydianion and orotate binding domains of OMPDC perform complementary functions in catalysis of decarboxylation reactions: (1) The orotate binding domain carries out decarboxylation of the orotate ring. (2) The activating oxydianion binding domain has the cryptic function of utilizing binding interactions with tetrahedral inorganic oxydianions to drive an enzyme conformational change that results in the stabilization of transition states at the distant orotate domain.

  14. Molecular characterization of Mtb-OMP decarboxylase by modeling, docking and dynamic studies. (United States)

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


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

  15. Nucleotide variation at the dopa decarboxylase (Ddc) gene in natural populations of Drosophila melanogaster

    Indian Academy of Sciences (India)

    Andrey Tatarenkov; Francisco J. Ayala


    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 -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.

  16. Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice.

    Directory of Open Access Journals (Sweden)

    Corette J Wierenga

    Full Text Available The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2% or vasoactive intestinal peptide (VIP; 15±2%, while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping expression of neuropeptide Y (42±3%, cholecystokinin (25±3%, calbindin (20±2% or calretinin (20±2%. Most of these subtypes (with the exception of calretinin-expressing interneurons target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.

  17. Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening. (United States)

    Rastogi, R; Dulson, J; Rothstein, S J


    Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from oat and Escherichia coli. Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained. This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein. Using this fragment as a probe, we isolated several partial ADC cDNA clones from a tomato pericarp cDNA library. The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene. The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD. The predicted amino acid sequence exhibits 47 and 38% identify with oat and E. coli ADCs, respectively. Gel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues. During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage. No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage. The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/or posttranslational regulation of ADC gene expression during tomato fruit ripening.

  18. Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus induces distinct behavioral deficits relevant to schizophrenia. (United States)

    Nguyen, Robin; Morrissey, Mark D; Mahadevan, Vivek; Cajanding, Janine D; Woodin, Melanie A; Yeomans, John S; Takehara-Nishiuchi, Kaori; Kim, Jun Chul


    Hyperactivity within the ventral hippocampus (vHPC) has been linked to both psychosis in humans and behavioral deficits in animal models of schizophrenia. A local decrease in GABA-mediated inhibition, particularly involving parvalbumin (PV)-expressing GABA neurons, has been proposed as a key mechanism underlying this hyperactive state. However, direct evidence is lacking for a causal role of vHPC GABA neurons in behaviors associated with schizophrenia. Here, we probed the behavioral function of two different but overlapping populations of vHPC GABA neurons that express either PV or GAD65 by selectively inhibiting these neurons with the pharmacogenetic neuromodulator hM4D. We show that acute inhibition of vHPC GABA neurons in adult mice results in behavioral changes relevant to schizophrenia. Inhibiting either PV or GAD65 neurons produced distinct behavioral deficits. Inhibition of PV neurons, affecting ∼80% of the PV neuron population, robustly impaired prepulse inhibition of the acoustic startle reflex (PPI), startle reactivity, and spontaneous alternation, but did not affect locomotor activity. In contrast, inhibiting a heterogeneous population of GAD65 neurons, affecting ∼40% of PV neurons and 65% of cholecystokinin neurons, increased spontaneous and amphetamine-induced locomotor activity and reduced spontaneous alternation, but did not alter PPI. Inhibition of PV or GAD65 neurons also produced distinct changes in network oscillatory activity in the vHPC in vivo. Together, these findings establish a causal role for vHPC GABA neurons in controlling behaviors relevant to schizophrenia and suggest a functional dissociation between the GABAergic mechanisms involved in hippocampal modulation of sensorimotor processes.

  19. Systematic analysis of γ-aminobutyric acid (GABA) metabolism and function in the social amoeba Dictyostelium discoideum. (United States)

    Wu, Yuantai; Janetopoulos, Chris


    While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several "early" developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development.

  20. Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hai-Yan Hu; Bing Zhang; Xian-Xi Liu; Chun-Ying Jiang; Yi Lu; Shi-Lian Liu; Ji-Feng Bian; Xiao-Ming Wang; Zhao Geng; Yan Zhang


    AIM: To investigate the ornithine decarboxylase (ODC)gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology.METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR.ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay.RESULTS: A cell line, which could steadily secrete antiODC mAb, was selected through subcloning four times.Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6±1.03%vs 5.26±5%, P<0.01).CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

  1. Expression of ornithine decarboxylase in precancerous and cancerous gastric lesions

    Institute of Scientific and Technical Information of China (English)

    Xin-Pu Miao; Jian-Sheng Li; Hui-Yan Li; Shi-Ping Zeng; Ye Zhao; Jiang-Zheng Zeng


    AIM:To investigate the expression of ornithine decarboxylase (ODC) in precancerous and cancerous gastric lesions.METHODS: We studied the expression of ODC in gastric mucosa from patients with chronic superficial gastritis (CSG, n = 32), chronic atrophic gastritis [CAG, n = 43;15 with and 28 without intestinal metaplasia (IM)],gastric dysplasia (DYS, n = 11) and gastric cancer (GC,n = 48) tissues using immunohistochemical staining. All 134 biopsy specimens of gastric mucosa were collected by gastroscopy.METHODS: The positive rate of ODC expression was 34.4%, 42.9%, 73.3%, 81.8% and 91.7% in cases with CSG, CAG without IM, CAG with IM, DYS and GC, respectively (P < 0.01), The positive rate of ODC expression increased in the order of CSG < CAG (without IM) < CAG (with IM) < DYS and finally, GC. In addition,ODC positive immunostaining rate was lower in welldifferentiated GC than in poorly-differentiated GC (P <0.05).CONCLUSION: The expression of ODC is positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. This finding indicates that ODC may be used as a good biomarker in the screening and diagnosis of precancerous lesions.

  2. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Kanerva, Kristiina; Maekitie, Laura T. [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); Baeck, Nils [Department of Anatomy, Institute of Biomedicine, University of Helsinki, Helsinki (Finland); Andersson, Leif C., E-mail: [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); HUSLAB, Helsinki (Finland); Department of Oncology and Pathology, Karolinska Institutet, Stockholm (Sweden)


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

  3. Structural Basis for Putrescine Activation of Human S-Adenosylmethionine Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Bale, Shridhar; Lopez, Maria M.; Makhatadze, George I.; Fang, Qingming; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Penn)


    Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

  4. Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.). (United States)

    Chang, K S; Lee, S H; Hwang, S B; Park, K Y


    Arginine decarboxylase (ADC; EC is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

  5. On-line near-infrared spectroscopy optimizing and monitoring biotransformation process of γ-aminobutyric acid

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    Guoyu Ding


    Full Text Available Near-infrared spectroscopy (NIRS with its fast and nondestructive advantages can be qualified for the real-time quantitative analysis. This paper demonstrates that NIRS combined with partial least squares (PLS regression can be used as a rapid analytical method to simultaneously quantify l-glutamic acid (l-Glu and γ-aminobutyric acid (GABA in a biotransformation process and to guide the optimization of production conditions when the merits of NIRS are combined with response surface methodology. The high performance liquid chromatography (HPLC reference analysis was performed by the o-phthaldialdehyde pre-column derivatization. NIRS measurements of two batches of 141 samples were firstly analyzed by PLS with several spectral pre-processing methods. Compared with those of the HPLC reference analysis, the resulting determination coefficients (R2, root mean square error of prediction (RMSEP and residual predictive deviation (RPD of the external validation for the l-Glu concentration were 99.5%, 1.62 g/L, and 11.3, respectively. For the GABA concentration, R2, RMSEP, and RPD were 99.8%, 4.00 g/L, and 16.4, respectively. This NIRS model was then used to optimize the biotransformation process through a Box-Behnken experimental design. Under the optimal conditions without pH adjustment, 200 g/L l-Glu could be catalyzed by 7148 U/L glutamate decarboxylase (GAD to GABA, reaching 99% conversion at the fifth hour. NIRS analysis provided timely information on the conversion from l-Glu to GABA. The results suggest that the NIRS model can not only be used for the routine profiling of enzymatic conversion, providing a simple and effective method of monitoring the biotransformation process of GABA, but also be considered to be an optimal tool to guide the optimization of production conditions.

  6. On-line near-infrared spectroscopy optimizing and monitoring biotransformation process ofγ-aminobutyric acid$

    Institute of Scientific and Technical Information of China (English)

    Guoyu Ding; Yuanyuan Hou; Jiamin Peng; Yunbing Shen; Min Jiang; Gang Bai


    Near-infrared spectroscopy (NIRS) with its fast and nondestructive advantages can be qualified for the real-time quantitative analysis. This paper demonstrates that NIRS combined with partial least squares (PLS) regression can be used as a rapid analytical method to simultaneously quantify L-glutamic acid (L-Glu) andγ-aminobutyric acid (GABA) in a biotransformation process and to guide the optimization of production conditions when the merits of NIRS are combined with response surface methodology. The high performance liquid chromatography (HPLC) reference analysis was performed by the o-phthaldialdehyde pre-column derivatization. NIRS measurements of two batches of 141 samples were firstly analyzed by PLS with several spectral pre-processing methods. Compared with those of the HPLC reference analysis, the resulting determination coefficients (R2), root mean square error of prediction (RMSEP) and residual predictive deviation (RPD) of the external validation for the L-Glu concentration were 99.5%, 1.62 g/L, and 11.3, respectively. For the GABA concentration, R2, RMSEP, and RPD were 99.8%, 4.00 g/L, and 16.4, re-spectively. This NIRS model was then used to optimize the biotransformation process through a Box-Behnken experimental design. Under the optimal conditions without pH adjustment, 200 g/L L-Glu could be catalyzed by 7148 U/L glutamate decarboxylase (GAD) to GABA, reaching 99%conversion at the fifth hour. NIRS analysis provided timely information on the conversion from L-Glu to GABA. The results suggest that the NIRS model can not only be used for the routine profiling of enzymatic conversion, providing a simple and effective method of monitoring the biotransformation process of GABA, but also be considered to be an optimal tool to guide the optimization of production conditions.

  7. Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2

    Directory of Open Access Journals (Sweden)

    Bruno Ramos-Molina


    Full Text Available Ornithine decarboxylase (ODC is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs, small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2 are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

  8. Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes. (United States)

    Stivers, J T; Washabaugh, M W


    Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide: pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha 4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C isotope discrimination against [1,2-14C]acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.

  9. Expression of GAD67 mRNA on epliptic rats kindled by electical amygdala%杏仁核点燃癫痫鼠GAD67 mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    林卫红; 张淑琴; 孟红梅; 寇玉红; 崔俐


    目的 探讨大鼠杏仁核点燃癫痫后脑组织谷氨酸脱羧酶(GAD)mRNA的表达及其在癫痫发作后表达变化的意义. 方法 通过建立与人类癫痫发生、形成具有高度相似性的杏仁核点燃大鼠癫痫模型, 采用原位杂交技术检测癫痫鼠颞叶及海马组织不同点燃时程GAD67 mRNA表达.结果 点燃后1d,GAD67 mRNA表达增多并且表达信号增强,至7d时达高峰,以后表达逐渐下降,但在点燃后49d,表达仍高于正常,与对照组及手术对照组相比有统计学差异. 结论 在慢性癫痫发作中,GABA能神经元的活性增强,考虑是由于癫痫过程中兴奋性增强,引起GABA能神经元抑制功能代偿性增加的结果 .即癫痫发作后GABA能神经元介入的抑制功能代偿性增加的,这可能是机体内源性抗癫痫机制增强的一种反应.

  10. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili


    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  11. Chilling Tolerance of Cucumber During Germination is Related to Expression of Lysine Decarboxylase Gene

    Institute of Scientific and Technical Information of China (English)

    LU Ming-hui; LI Xiao-ming; CHEN Jin-feng; CHEN Long-zheng; QIAN Chun-tao


    Using cDNA-AFLP technique, a specific fragment was isolated from cucumber cultivar Changchun mici possessing chilling tolerance induced at low temperature (15℃). This fragment, named cctr 132, could not be induced in the chilling sensitive cucumber cultivar Beijing jietou. After recovering the fragment, sequencing and translating, the results of blastx and blastp in GenBank of NCBI indicated that CCTR132 had 88.37% identities and 100% positives with Oryza sativa putative lysine decarboxylase-like protein respectively, and PGGXGTXXE, the putative conserved domain of lysine decarboxylase family, was detected from CCTR132, suggesting the cucumber chilling tolerance during germination is related to the expression of the lysine decarboxylase gene.

  12. Paleomagnetism of Late Paleozoic series in Morocco and Argentina: implications for GAD Hypothesis and Pangea reconstruction (United States)

    Besse, J.; Oufi, O.; Rapalini, A.; Courtillot, V. E.


    The configuration of Pangea during the upper Paleozoic is still strongly debated: the APWP for Gondwana and Laurussia should fit in the GAD hypothesis when Pangea is reconstructed using marine data. But they appear to be significantly distinct, by as much as 15° for certain periods. Motion between the two blocks has been proposed (B Pangea of Irving) but is geologically problematic. Erroneous age assignments, magnetic overprints, insufficient demagnetization, problems with the recording of geomagnetic field in sediments (f.i. flattening) and finally non-dipole contributions have all been invoked to reconcile apparently discrepant poles. In this talk, we investigate the geometry of the geomagnetic field during the Late Carboniferous-Early Triassic period (320-240 Ma). As a starting point, we use two recent surveys in Morocco and Argentina, which provide paleomagnetic tests and good age control that were missing in most previous studies and yield respectively paleolatitude data close to the equator and mid/high latitudes. Using a compilation of poles from the GPMDB, we analyze the positions of mean poles for mid-northern and southern, and equatorial latitudes, searching for the distinctive antisymmetrical pattern expected for a dipole with an octupole contribution. We also discuss the main causes of errors, such as the occurrence of lithospheric deformation, which induces important rotations at various scales, particularly in future rift or mountain zones (Colorado, South of France, South American cordilleras, east of Australia, etc.). Even when using only sampling sites close to the Paleo-equator (which minimizes any octupolar effect), the APWP of Gondwana remains offset from that of Laurussia. The plate configuration inferred is a classical A Pangea reconstruction at about 260 Ma, but data do not rule out the possibility of a B Pangea before 270 Ma, which would account for a large number of geological constraints. In contradiction with recently proposed persistent

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

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    Melissa Gamat

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

  14. Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii. (United States)

    Lu, Zichun J; Markham, George D


    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.

  15. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)


    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.

  16. Combination therapy with an anti-IL-1β antibody and GAD65 DNA vaccine can reverse recent-onset diabetes in the RIP-GP mouse model. (United States)

    Pagni, Philippe P; Bresson, Damien; Rodriguez-Calvo, Teresa; Bel Hani, Amira; Manenkova, Yulia; Amirian, Natalie; Blaszczak, Alecia; Faton, Sina; Sachithanantham, Sowbarnika; von Herrath, Matthias G


    Type 1 diabetes is thought to be an autoimmune condition in which self-reactive T cells attack insulin-secreting pancreatic β-cells. As a proinflammatory cytokine produced by β-cells or macrophages, interleukin-1β (IL-1β) represents a potential therapeutic target in diabetes. We reasoned IL-1β blockade could be combined with islet antigen-specific approaches involving GAD of 65 kDa (GAD65)-expressing plasmids, as previously shown in combination therapies (CTs) with anti-CD3. Thus, we investigated whether anti-IL-1β antibody alone or combined with GAD65 vaccine could reverse diabetes development in a virus-induced mouse model. Given alone, anti-IL-1β had no effect on diabetes, while GAD65 plasmid resulted in 33% disease reversal after a 5-week observation. However, CTs cured 53% of animals and prevented worsening of glycemic control in nonprotected individuals for up to 12 weeks. While the GAD65 vaccine arm of the CT was associated with increased forkhead box p3(+) regulatory T-cell frequency in pancreatic lymph nodes, islet infiltration by CD11b(+/high) cells was less frequent upon CT, and its extent correlated with treatment success or failure. Altogether, our CTs provided prolonged improvement of clinical and immunological features. Despite unsuccessful clinical trials using anti-IL-1β monotherapy, these data hold promise for treatment of type 1 diabetic patients with IL-1β blockade combined with antigen-specific vaccines.

  17. Expression of GAD of cochlea in FMR1 gene Knock-out mice%Fmr1基因敲除小鼠耳蜗GAD的表达

    Institute of Scientific and Technical Information of China (English)

    李敏雄; 杜娜; 孙卫文; 黄月玲; 沈岩松; 戴丽军; 陈盛强; 马钊恩; 张建国


    目的 对4 周龄Fmr1 基因敲除小鼠的耳蜗的GAD 表达进行观察,探讨耳蜗GAD 的表达是否受FMRP 的影响.方法 使用PCR 技术对Fmr1 基因敲除小鼠鉴定后对4 周龄的Fmr1 基因敲除小鼠和野生型小鼠各15 只进行耳蜗的苏木精-伊红(HE) 染色和GAD 免疫组织化学的表达观察,数据采用多因素方差分析处理.结果 耳蜗HE 染色结果:4 周龄组KO 鼠较WT 鼠形态学观察无差异.4 周龄KO 小鼠的耳蜗中GAD 表达的平均阳性细胞数均高于WT 小鼠,P<0.01,差异具有统计学意义.结论 GAD 表达的改变可能与FMR1 基因KO 小鼠听源性惊厥发病有关.

  18. No Contribution of GAD-65 and IA-2 Autoantibodies around Time of Diagnosis to the Increasing Incidence of Juvenile Type 1 Diabetes: A 9-Year Nationwide Danish Study

    Directory of Open Access Journals (Sweden)

    Steffen U. Thorsen


    Full Text Available Aims. A new perspective on autoantibodies as pivotal players in the pathogenesis of type 1 diabetes (T1D has recently emerged. Our key objective was to examine whether increased levels of autoantibodies against the β-cell autoantigens glutamic acid decarboxylase (isoform 65 (GADA and insulinoma associated antigen-2A (IA-2A mirrored the 3.4% annual increase in incidence of T1D. Methods. From the Danish Childhood Diabetes Register, we randomly selected 500 patients and 500 siblings for GADA and IA-2A analysis (1997 through 2005. Blood samples were taken within three months after onset. A robust log-normal regression model was used. Nine hundred children and adolescents had complete records and were included in the analysis. Cochran-Armitage test for trend was used to evaluate changes in prevalence of autoantibody positivity by period. Results. No significant changes in levels of GADA and IA-2A were found over our 9-year study period. No trends in autoantibody positivity—in either patients or siblings—were found. Levels of GADA and IA-2A were significantly associated with HLA risk groups and GADA with age. Conclusion. The prevalence of positivity and the levels of GADA and IA-2A have not changed between 1997 and 2005 in newly diagnosed patients with T1D and their siblings without T1D.

  19. Long-lasting c-fos and NGF mRNA expressions and loss of perikaryal parvalbumin immunoreactivity in the development of epileptogenesis after ethacrynic acid-induced seizure. (United States)

    Suzukawa, J; Omori, K; Okugawa, G; Fujiseki, Y; Heizmann, C W; Inagaki, C


    A single cerebroventricular injection of ethacrynic acid (EA), a Cl(-)-ATPase inhibitor, induces generalized tonic-clonic convulsions in mice. To clarify whether such convulsive stimulus triggers a long-lasting rearrangement of the neural circuitry culminating in seizure susceptibility, we examined molecular, cellular and behavioral changes following the EA-induced seizure. The expression of immediate early gene c-fos mRNA as an index for cellular activation increased biphasically, with an early transient increase at 60 min and a late prolonged increase on the 10th to 14th day post-EA administration, most remarkably in the hippocampus and pyriform cortex. On the 14th day post-EA seizure, subconvulsive dose of kainic acid (5-17.5 mg/kg) caused severe (stage 5) seizure in 77% of the mice, with 70% mortality. In addition, the expression of nerve growth factor (NGF) also showed biphasic increases with close spatiotemporal correlation with c-fos expression. Moreover, the number of cell somata and the density of axon fibers of parvalbumin (PARV)-positive cells, a subpopulation of GABAergic interneurons, decreased in area dentata, CA1 and CA3 on the 7th and 14th day post-EA seizure. In area dentata and CA1, the density of glutamic acid decarboxylase (GAD)-positive cells also decreased on the 14th day. Thus, the transient EA-induced seizures appear to develop seizure susceptibility by causing damage of a subpopulation of inhibitory interneurons along with increases in the expression of c-fos and NGF in limbic structures.

  20. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity. (United States)

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y


    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

  1. Glucocorticoids modulate the response of ornithine decarboxylase to unilateral removal of the dorsal hippocampus

    NARCIS (Netherlands)

    De Kloet, E R; Cousin, M A; Veldhuis, H D; Voorhuis, T D; Lando, D


    The effect of unilateral removal of the dorsal hippocampus and of glucocorticoid administration was measured on the activity of ornithine decarboxylase (ODC) in the remaining contralateral hippocampus lobe. Unilateral hippocampectomy (Hx) resulted in a rapid rise of ODC activity in the contralateral

  2. Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum

    NARCIS (Netherlands)

    Stekelenburg, Gerard J. van; Koorevaar, Gerrit


    In this article evidence is presented for the existence of mammalian acetoacetate decarboxylase (acetoacetate carboxy-lyase: E.G. From experiments with human blood serum the presence of a non-ultrafiltrable activator, accelerating the decomposition of acetoacetate into acetone and carbon d

  3. Polyamine biosynthesis and degradation are modulated by exogenous gamma-aminobutyric acid in root-zone hypoxia-stressed melon roots. (United States)

    Wang, Chunyan; Fan, Longquan; Gao, Hongbo; Wu, Xiaolei; Li, Jingrui; Lv, Guiyun; Gong, Binbin


    We detected physiological change and gene expression related to PA metabolism in melon roots under controlled and hypoxic conditions with or without 5 mM GABA. Roots with hypoxia treatment showed a significant increase in glutamate decarboxylase (GAD) activity and endogenous GABA concentration. Concurrently, PA biosynthesis and degradation accelerated with higher gene expression and enzymes activity. However, endogenous GABA concentrations showed a large and rapid increase in Hypoxia + GABA treated roots. This led to a marked increase in Glu concentration by feedback inhibition of GAD activity. Hypoxia + GABA treatment enhanced arginine (Arg), ornithine (Orn) and methionine (Met) levels, promoting enzyme gene expression levels and arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities in roots. Hypoxia + GABA treatment significantly increased concentrations of free putrescine (Put), spermidine (Spd) and spermine (Spm) from day two to eight, promoting the PA conversion to soluble conjugated and insoluble bound forms. However, PA degradation was significantly inhibited in hypoxia + GABA treated roots by significantly decreasing gene expression and activity of diamine oxidase (DAO) and polyamine oxidase (PAO). However, exogenous GABA showed a reduced effect in control compared with hypoxic conditions. Our data suggest that alleviating effect of exogenous GABA to hypoxia is closely associated with physiological regulation of PA metabolism. We propose a potential negative feedback mechanism of higher endogenous GABA levels from combined effects of hypoxia and exogenous GABA, which alleviate the hypoxia damage by accelerating PA biosynthesis and conversion as well as preventing PA degradation in melon plants.

  4. 氯化盐和激素对发芽蚕豆中γ-氨基丁酸富集的影响%Effects of chloride salts and hormones on γ-aminobutyric acid enrichment in germinated faba beans( Vicia faba L.)

    Institute of Scientific and Technical Information of China (English)

    陈惠; 杨润强; 李岩; 韩永斌; 顾振新


    Effects of CaCl2,NaCl,gibberellic acid(GA3)and abscisic acid (ABA) on glutamate decarboxylase(GAD) ,diamine oxidases (DAO)and -γ-aminobutyric acid(GABA)of germinated faba beans ( Vicia faba L. ) , together with the contribution rate of polyamines (Pas) degradation to the enrichment of GABA were studied. The results showed that CaCl2, NaCl and GA3, except ABA, could increase the content of GABA in germinated faba beans under hypoxia stress. It was indicated that the optimal culture components for GABA en richment were:NaCl at a concentration of 37.1 mmol·L-1 ,CaCl2 at a concentration of 7. 3 mmol·L-1 and GA3 at a concentration of 27. 0 μmol·L-1. Under those conditions,the content of GABA was the highest,reaching(6. 14±0. 12) mg·g-1 ,which was 1. 58 times of the control. And the contribution rate of Pas degradation to enrichment of GABA ranged from 37.6% to 38.9% .%以蚕豆(Vicia faba L.)为试材,研究CaCl2 、NaCl、赤霉素(GA3)和脱落酸(ABA)对发芽蚕豆谷氨酸脱羧酶(GAD)、二胺氧化酶(DAO)和γ-氨基丁酸(GABA)的影响,以及多胺(PAs)降解对GABA富集的贡献率.结果表明:低氧胁迫下,除ABA外,CaCl2、NaCl和GA3处理均可提高发芽蚕豆GABA含量;培养液组分中NaCl、CaCl2、GA3浓度分别为37.1 mmol·L-1、7.3 mmol·L-1和27.0 μmol·L-1时,发芽蚕豆中GABA富集量(6.14±0.12) mg·g-1是对照的1.58倍;PAs降解对GABA富集的贡献率为37.6%~ 38.9%.

  5. Pyridoxal 5'-phosphate (PLP) deficiency might contribute to the onset of type I diabetes. (United States)

    Rubí, B


    The incidence of type I diabetes is rising worldwide, particularly in young children. Type I diabetes is considered a multifactorial disease with genetic predisposition and environmental factors participating. Currently, despite years of research, there is no consensus regarding the factors that initiate the autoimmune response. Type I diabetes is preceded by autoimmunity to islet antigens, among them the protein glutamic acid decarboxylase, GAD-65. Pyridoxal 5'-phosphate (PLP) is formed from vitamin B6 by the action of pyridoxal kinase. Interaction of GAD65 with PLP is necessary for GAD65-mediated synthesis of the neurotransmitter γ-aminobutyric acid (GABA). PLP is also a required cofactor for dopamine synthesis by L-aromatic decarboxylase (L-AADC). Both GAD65 and L-AADC are expressed in pancreatic islets. Here it is proposed that lack of the vitamin B6 derivative pyridoxal 5'-phosphate might contribute to the appearance of pancreatic islet autoimmunity and type I diabetes onset.

  6. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods. (United States)

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara


    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  7. GABA and Glutamate are not colocalized in mossy fiber terminals of developing rodent hippocampus


    Xiong, Guoxiang; Zhang, Lei; Mojsilovic-Petrovic, Jelena; Arroyo, Edguardo; Elkind, Jaclynn; Kundu, Suhali; Johnson, Brian; Smith, Colin J.; Cohen, Noam A.; Grady, Sean M.; Cohen, Akiva S.


    It has been hypothesized that, in the developing rodent hippocampus, mossy fiber terminals release GABA together with glutamate. Here, we used transgenic glutamic acid decarboxylase-67 (GAD67)-GFP expressing mice and multi-label immunohistochemistry to address whether glutamatergic and GABAergic markers are colocalized. We demonstrate that in the dentate gyrus, interneurons positive for GABA/GAD are sparsely distributed along the edge of the hilus, in a different pattern than the densely pack...

  8. A dopa decarboxylase modulating the immune response of scallop Chlamys farreri.

    Directory of Open Access Journals (Sweden)

    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.

  9. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

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    Pohl Martina


    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  10. The Effects of Two-Week Swimming Training on Neuropathic Pain Induced by Chronic Constriction Injury and the Expression of GAD65 in Adult Male Rats

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    Babak Farzad


    Full Text Available Background & Objective: Unknown mechanisms are involved in neuropathic pain. Among the non-pharmacological treatments, it seems that physical activity improves neuropathic pain. However, the possible reasons for the effectiveness of regular physical activity on neuropathic pain are unknown. Therefore, the present study was performed to determine the effects of two-week swimming training on the expression of GAD65 enzyme and P2X3 receptor in Chronic Constriction Injury (CCI of the sciatic nerve. Materials & Methods: 40Wistar adult rats were divided into five groups randomly: 1 CCI neuropathic pain with swimming training (CCIST2; 2 CCI neuropathic pain without swimming training (CCI; 3 No CCI neuropathic pain with swimming training (ST2; No CCI neuropathic pain without swimming training (control group; 5 CCI sham surgery (Sham CCI. CCI and CCIST2 groups underwent peripheral nerve injury by four loose ligatures around sciatic nerve. Swimming program included two weeks with five sessions per week, and 30-60 min per session. The protein expressions of GAD65 enzyme and P2X3 receptor were evaluated by western blotting technique. Results: CCI surgery decreased the expression of GAD65, but two weeks swimming training increased expression of GAD65 comparing to CCI and Sham CCI groups (P≤0.001, but P2X3 receptor expression were not significantly different among groups in lumbar segment of rats (P>0.05. Conclusion: Totally, our findings showed that two-week swimming training improves neuropathic pain possibly through maintenance of inhibitory neurons and subsequently increased GAD65, which converts glutamate excitatory neurotransmitter to GABA inhibitory neurotransmitter.

  11. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    Energy Technology Data Exchange (ETDEWEB)

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.


    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  12. Increase of histidine decarboxylase activity in mice hypothalamus after intracerebroventricular administration of lipopolysaccharide. (United States)

    Niimi, M; Mochizuki, T; Cacabelos, R; Yamatodani, A


    The effect of intracerebroventricular (icv) administration of lipopolysaccharide on histidine decarboxylase activity and histamine content in the hypothalamus were investigated in male mice of ddY strain in vivo. Two-fold increase in histidine decarboxylase activity (HDC) was observed 4 h after administration of 50 mcg lipopolysaccharide, and HDC activity returned to the basal level within 12 h after injection. Furthermore, histamine contents showed a slight decrease at 1 and 2 h and a mild increase at 12 h after administration. However, changes in histamine content were not statistically significant. These results suggest that the increase of HDC activity in the hypothalamus by lipopolysaccharide may be involved in the central neuroimmune responses.

  13. Autoimmune disease

    Institute of Scientific and Technical Information of China (English)


    2005164 Optimal cut-point of glutamic acid decar-boxylase antibody (GAD-Ab) for differentiating two subtypes of latent autoimmune diabetes in adults (LADA). LI Xia(李霞), et al. Dept Endocrinol, 2nd Xiangya Hosp, Central South Univ, Changsha, 410011. Chin J Diabetes, 2005;13(1) :34-38. Objective: To investigate the optimal cut-point of glutamate decarboxylase antibody (GAD-Ab) for differentiating two subtypes of latent autoimmune diabetes in adults (I. ADA). Methods: The frequency

  14. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica. (United States)

    Toutges, Michelle J; Santoso, Adi


    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene.

  15. Real-Time kinetic studies of Bacillus subtilis oxalate decarboxylase and Ceriporiopsis subvermispora oxalate oxidase using a luminescent oxygen sensor

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    Laura Molina


    Full Text Available Oxalate decarboxylase (OxDC, an enzyme of the bicupinsuperfamily, catalyzes the decomposition of oxalate into carbondioxide and formate at an optimal pH of 4.3 in the presence ofoxygen. However, about 0.2% of all reactions occur through anoxidase mechanism that consumes oxygen while producing twoequivalents of carbon dioxide and one equivalent of hydrogenperoxide. The kinetics of oxidase activity were studied bymeasuring the consumption of dissolved oxygen over time using a luminescent oxygen sensor. We describe the implementation of and improvements to the oxygen consumption assay. The oxidase activity of wild type OxDC was compared to that of the T165V OxDC mutant, which contains an impaired flexible loop covering the active site. The effects of various carboxylic acid-based buffers on the rate of oxidase activity were also studied. These results were compared to the oxidase activity of oxalate oxidase (OxOx, a similar bicupin enzyme that only carries out oxalate oxidation. Thetemperature dependence of oxidase activity was analyzed, andpreliminary results offer an estimate for the overall activationenergy of the oxidase reaction within OxDC. The data reported here thus provide insights into the mechanism of the oxidase activity of OxDC.

  16. The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes. (United States)

    Sykes, Steven; Szempruch, Anthony; Hajduk, Stephen


    α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

  17. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis. (United States)

    Mergler, M; Klinner, U


    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  18. Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase. (United States)

    Harris, T K; Washabaugh, M W


    The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from Leishmania donovani.

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    Saurabh Pratap Singh

    Full Text Available In addition to the S-adenosylmethionine decarboxylase (AD present in all organisms, trypanosomatids including Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL. Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.

  20. Reduced by-product formation and modified oxygen availability improve itaconic acid production in Aspergillus niger.

    NARCIS (Netherlands)

    Li, A.; Pfelzer, N.; Zuijderwijk, R.; Brickwedde, A.; Zeijl, C. van; Punt, P.


    Aspergillus niger has an extraordinary potential to produce organic acids as proven by its application in industrial citric acid production. Previously, it was shown that expression of the cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus converted A. niger into an itaconic acid produ

  1. The ornithine decarboxylase, NO-synthase activities and phospho-c-Jun content under experimental gastric mucosa malignancy

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    Mariia Tymoshenko


    Full Text Available Ornithine decarboxylase is the first and key regulatory enzyme in synthesis of polyamines, which are essential for cell proliferation and differentiation, so its aberrant regulation is reported to play a role in neoplastic transformation and tumours growth. That's why, there were analysed some major links of metabolic pathways that are closely related to tumorigenesis: ornithine decarboxylase, and the NADPH-dependent enzyme nitric oxide synthase, the nuclear phosphoprotein c-Jun, that could play an important role in the development of gastric cancer malignancy.The gastric carcinogenesis was initiated in rats by 10-week replacement of drinking water by 0.01% N-methyl-N-nitro-N-nitrosoguanidine solution, at the same time they were redefined on the diet containing 5% NaCl. After this period expiry the animals were fed with standard diet till the end of the 24th week. The gastric mucosa cells were extracted at the end of the 4th, 6th, 8th, 10th, 12th, 18th and 24th week and underwent biochemical examinations. It was established the elevated phospho-c-Jun content, ornithine decarboxylase and inducible nitric oxide synthase activities from 6th to 24th week of gastric cancer development compared to the control references. The increasing of ornithine decarboxylase activity could probably be caused by the growth of phospho-c-Jun, it is also belonging to an ornithine decarboxylase transactivation effects. Thus, it was shown that the increase of ornithine decarboxylase and inducible nitric oxide synthase activities, phospho-c-Jun and nitrite-ions accumulation in gastric mucosa epithelial cells were associated with the gastric malignant progression. The complex relationships between the examined enzymes and transcription activator that pointed to an aggravation of pathological disturbances due to reciprocal action between ornithine decarboxylase and c-Jun and nitric oxide synthase participation. [Biomed Res Ther 2016; 3(4.000: 596-604

  2. Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina (United States)

    Ishida, Yuko; Ozaki, Mamiko


    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.


    Institute of Scientific and Technical Information of China (English)


    The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC)were studied This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the efffect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.O ×1O-5 M. The linearity response is in the range of 5 × 1O -2-5 × 1O -5 M. The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

  4. Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model (United States)

    Subburaju, S; Coleman, A J; Ruzicka, W B; Benes, F M


    Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ. PMID:26812044

  5. Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model. (United States)

    Subburaju, S; Coleman, A J; Ruzicka, W B; Benes, F M


    Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ.

  6. The direction of effects between perceived parental behavioral control and psychological control and adolescents' self-reported GAD and SAD symptoms


    Wijsbroek, Saskia A. M.; Hale III, William W.; Raaijmakers, Quinten A. W.; Meeus, Wim H. J.


    Abstract This study examined the direction of effects and age and sex differences between adolescents? perceptions of parental behavioral and psychological control and adolescents? self-reports of generalized anxiety disorder (GAD) and separation anxiety disorder (SAD) symptoms. The study focused on 1,313 Dutch adolescents (early-to-middle cohort n = 923, 70.3%; middle-to-late cohort n = 390, 29.7%) from the general population. A multi-group, structural equation model was employed ...

  7. Development of a Novel Cysteine Sulfinic Acid Decarboxylase Knockout Mouse: Dietary Taurine Reduces Neonatal Mortality

    Directory of Open Access Journals (Sweden)

    Eunkyue Park


    Full Text Available We engineered a CSAD KO mouse to investigate the physiological roles of taurine. The disruption of the CSAD gene was verified by Southern, Northern, and Western blotting. HPLC indicated an 83% decrease of taurine concentration in the plasma of CSAD-/-. Although CSAD-/- generation (G1 and G2 survived, offspring from G2 CSAD-/- had low brain and liver taurine concentrations and most died within 24 hrs of birth. Taurine concentrations in G3 CSAD-/- born from G2 CSAD-/- treated with taurine in the drinking water were restored and survival rates of G3 CSAD-/- increased from 15% to 92%. The mRNA expression of CDO, ADO, and TauT was not different in CSAD-/- compared to WT and CSAD mRNA was not expressed in CSAD-/-. Expression of Gpx 1 and 3 was increased significantly in CSAD-/- and restored to normal levels with taurine supplementation. Lactoferrin and the prolactin receptor were significantly decreased in CSAD-/-. The prolactin receptor was restored with taurine supplementation. These data indicated that CSAD KO is a good model for studying the effects of taurine deficiency and its treatment with taurine supplementation.

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

    DEFF Research Database (Denmark)

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


    a loss of inhibition by descending 5-HT neurons and to be mediated by 5-HT1B receptors expressed by AADC cells. These findings indicate that AADC cells are a potential source of 5-HT at spinal levels below an SCI. The production of 5-HT by AADC cells, together with an upregulation of 5-HT2 receptors...

  9. ¹H, ¹³C and ¹⁵N resonance assignments and second structure information of Gad m 1: a β-parvalbumin allergen from Atlantic cod (Gadus morhua). (United States)

    Moraes, A H; Ackerbauer, D; Kostadinova, M; Bublin, M; Ferreira, F; Almeida, F C L; Breiteneder, H; Valente, A P


    Gad m 1 is the major allergen from Atlantic cod. It belongs to β-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. β-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of β-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a β-parvalbumin: Gad m 1. This report includes: (1)H, (13)C and (15)N resonance assignments of Gad m 1 as well as the second structure information based on the (13)C chemical shifts.

  10. Acquisition of a heat stable enzyme; S-adenosylmethionine decarboxylase from selenomonas ruminantium

    Energy Technology Data Exchange (ETDEWEB)

    Ko, Kyong Cheol; Park, Sang Hyun [Radiation Research Center for Biotechnology, 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)


    In Selenomoans ruminantium, a strictly anaerobic and gram negative bacterium, cadaverine and putrescine are the essential constituents of its peptidoglycan. S. ruminantium does not contain both free and bound types of lipoprotein, but it contains cadaverine as a component of its peptidoglycan. S-adenosylmethionine decarboxylase (SAMDC) is a key enzyme for a synthesis of spermidine and spermine in S. ruminantium. The crude extract of S. ruminantium was preincubated at 100 degrees Celcius and its SAMDC activity was measured by using a {sup 14}C labeled substrate. We report here on a heat stable SAMDC which is able to withstand a temperature up to 100 degrees Celcius.

  11. Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography. (United States)

    Haraguchi, K; Kai, M; Kohashi, K; Ohkura, Y


    A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

  12. The response of GABAergic and cholinergic neurons to transient cerebral ischemia. (United States)

    Francis, A; Pulsinelli, W


    The vulnerability of striatal and hippocampal neurons to ischemia was studied by measuring the activity of neurotransmitter-related enzymes after transient forebrain ischemia in rats. Activities of glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) were measured 6 h to 8 days after 20, 30 or 40 min of forebrain ischemia, as markers for GABAergic and cholinergic neurons respectively. Transient forebrain ischemia resulted in depression of striatal GAD activity while striatal CAT and hippocampal GAD activities were unaffected. Striatal GAD activity progressively decreased during the first 24 h postischemia and remained depressed 5--8 days later, suggesting irreversible damage to this population of neurons. The stability of striatal CAT and hippocampal GAD activity indicates that these cells were resistant to the present ischemic conditions.

  13. [Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale]. (United States)

    Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Guo, Shun-Xing


    S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

  14. Secondary. beta. -deuterium isotope effects in decarboxylation and elimination reactions of. cap alpha. -lactylthiamin: intrinsic isotope effects of pyruvate decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Kluger, R.; Brandl, M.


    The reactions of the adduct of pyruvate and thiamine, lactylthiamin (2-(lact-2-yl)thiamine), are accurate nonenzymic models for reactions of intermediates formed during catalysis by pyruvate decarboxylase. The enzymatic reaction generates lactylthiamin diphosphate from pyruvate and thiamine diphosphate. ..beta..-Deuterium isotope effects were determined for the nonenzymic reactions, and the results were related to isotope effects on the enzymic reaction. 2-(Lact-2-yl-..beta..-d/sub 3/) thiamine was prepared by condensation of methyl pyruvate-d/sub 3/ with thiamine followed by hydrolysis. The isotope effect for decarboxylation of lactylthiamin in acidic solution at 25/sup 0/C (k/sub H3//k/sub D3/) is 1.09 (standard deviation (SD) 0.015) in pH 3.8, 0.5 M sodium acetate: isotope effect = 1.095 (SD 0.014) in 0.001 M HCl. The reaction was also studied using 38% ethanolic aqueous sodium acetate (pH 3.8 before mixing with ethanol) since the enzymic sites are less polar than water and the reaction is significantly accelerated by the cosolvent. The isotope effect is within statistical range of that for the reaction in water, 1.105 (SD 0.016), indicating that acceleration by the solvent does not change the extent of hyperconjugative stabilization of the transition state relative to the ground state. The isotope effect for the base-catalyzed elimination of pyruvate from lactylthiamin was determined from kinetic studies by using multiwavelength analysis for reactions in pH 11 sodium carbonate solution. The isotope effect (k/sub H3//k/sub D3/) is 1.12 (SD 0.01), which is slightly higher than the effect on decarboxylation.

  15. Decarboxylase gene expression and cadaverine and putrescine production by Serratia proteamaculans in vitro and in beef. (United States)

    De Filippis, Francesca; Pennacchia, Carmela; Di Pasqua, Rosangela; Fiore, Alberto; Fogliano, Vincenzo; Villani, Francesco; Ercolini, Danilo


    Studies of the molecular basis of microbial metabolic activities that are important for the changes in food quality are valuable in order to help in understanding the behavior of spoiling bacteria in food. The growth of a psychrotrophic Serratia proteamaculans strain was monitored in vitro and in artificially inoculated raw beef. Two growth temperatures (25°C and 4°C) were tested in vitro, while growth at 15°C and 4°C was monitored in beef. During growth, the expression of inducible lysine and ornithine-decarboxylase genes was evaluated by quantitative reverse transcription-PCR (qRT-PCR), while the presence of cadaverine and putrescine was quantified by LC-ESI-MS/MS. The expression of the decarboxylase genes, and the consequent production of cadaverine and putrescine were shown to be influenced by the temperature, as well as by the complexity of the growth medium. Generally, the maximum gene expression and amine production took place during the exponential and early stationary phase, respectively. In addition, lower temperatures caused slower growth and gene downregulation. Higher amounts of cadaverine compared to putrescine were found during growth in beef with the highest concentrations corresponding to microbial loads of ca. 9CFU/g. The differences found in gene expression evaluated in vitro and in beef suggested that such activities are more reliably investigated in situ in specific food matrices.

  16. Rational design of ornithine decarboxylase with high catalytic activity for the production of putrescine. (United States)

    Choi, Hyang; Kyeong, Hyun-Ho; Choi, Jung Min; Kim, Hak-Sung


    Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes.

  17. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats (United States)

    Hoffman, Gloria E.; Koban, Michael


    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. PMID:27997552

  18. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues. (United States)

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P


    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

  19. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae. (United States)

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki


    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

  20. Evolutionary trails of plant group II Pyridoxal phosphate-dependent decarboxylase genes

    Directory of Open Access Journals (Sweden)

    Rahul Kumar


    Full Text Available 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 (HDC 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.

  1. Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

    Energy Technology Data Exchange (ETDEWEB)

    Abell, L.M.; O' Leary, M.H.


    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9770 +/- 0.0021, a carbon isotope effect k/sup 12//k/sup 13/ = 1.0308 +/- 0.0006, and a carbon isotope effect for L-(..cap alpha..-/sup 2/H)histidine of 1.0333 +/- 0.0001 at pH 6.3, 37/sup 0/C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

  2. Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase from Alcaligenes bronchisepticus

    Energy Technology Data Exchange (ETDEWEB)

    Nakasako, Masayoshi, E-mail: [Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); The RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Obata, Rika; Okubo, Ryosuke; Nakayama, Shyuichi [Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); Miyamoto, Kenji; Ohta, Hiromichi [Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan)


    Crystals of arylmalonate decarboxylase from A. bronchisepticus were obtained which diffracted X-rays to a resolution of at least 3.0 Å. Arylmalonate decarboxylase catalyses the enantioselective decarboxylation of α-aryl-α-methylmalonates to produce optically pure α-arylpropionates. The enzyme was crystallized with ammonium sulfate under alkaline pH conditions with the aim of understanding the mechanism of the enantioselective reaction. X-ray diffraction data collected to a resolution of 3.0 Å at cryogenic temperature showed that the crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 83.13, b = 99.62, c = 139.64 Å. This suggested that the asymmetric unit would contain between four and six molecules. Small-angle X-ray scattering revealed that the enzyme exists as a monomer in solution. Thus, the assembly of molecules in the asymmetric unit was likely to have been induced during the crystallization process.

  3. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus... (United States)


    ... preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173.115 Food and Drugs FOOD AND DRUG... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may be... derived from a modified Bacillus subtilis strain that contains the gene coding for α-ALDC from...

  4. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community. (United States)

    Zargar, K; Saville, R; Phelan, R M; Tringe, S G; Petzold, C J; Keasling, J D; Beller, H R


    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

  5. Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms. (United States)

    Goodman, S A; Esau, B; Koontz, J W


    Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

  6. Development of an Acid-Resistant Salmonella Typhi Ty21a Attenuated Vector For Improved Oral Vaccine Delivery (United States)

    Feuille, Catherine M.; Starke, Carly Elizabeth C.; Bhagwat, Arvind A.; Stibitz, Scott; Kopecko, Dennis J.


    The licensed oral, live-attenuated bacterial vaccine for typhoid fever, Salmonella enterica serovar Typhi strain Ty21a, has also been utilized as a vaccine delivery platform for expression of diverse foreign antigens that stimulate protection against shigellosis, anthrax, plague, or human papilloma virus. However, Ty21a is acid-labile and, for effective oral immunization, stomach acidity has to be either neutralized with buffer or by-passed with Ty21a in an enteric-coated capsule (ECC). Several studies have shown that efficacy is reduced when Ty21a is administered in an ECC versus as a buffered liquid formulation, the former limiting exposure to GI tract lymphoid tissues. However, the ECC was selected as a more practical delivery format for both packaging/shipping and vaccine administration ease. We have sought to increase Ty21a acid-resistance to allow for removal from the ECC and immune enhancement. To improve Ty21a acid-resistance, glutamate-dependent acid resistance genes (GAD; responsible for Shigella spp. survival at very low pH) were cloned on a multi-copy plasmid (pGad) under a controllable arabinose-inducible promoter. pGad enhanced acid survival of Ty21a by 5 logs after 3 hours at pH 2.5, when cells were pre-grown in arabinose and under conditions that promote an acid-tolerance response (ATR). For genetically 100% stable expression, we inserted the gad genes into the Ty21a chromosome, using a method that allowed for subsequent removal of a selectable antibiotic resistance marker. Further, both bacterial growth curves and survival assays in cultured human monocytes/macrophages suggest that neither the genetic methods employed nor the resulting acid-resistance conferred by expression of the Gad proteins in Ty21a had any effect on the existing attenuation of this vaccine strain. PMID:27673328

  7. Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification.

    Directory of Open Access Journals (Sweden)

    Changjiu Zhao

    Full Text Available Gamma-aminobutyric acid (GABA neurotransmission in the lateral septum (LS is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD, the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2 and GAD67 (GAD1, and used fluorescence in Situ hybridization (FISH with tyramide signal amplification (TSA to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg and medial preoptic area (MPOA in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%, while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%. Using the neuronal marker NeuN, almost every neuron in LS (> 90% was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB or calretinin (CR, but not parvalbumin (PV; almost all CB- or CR-immunoreactive neurons (98-100% were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI (approximately 58% for CB and 35% for CR. These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of

  8. Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss. (United States)

    Chen, G C; Jordan, F


    A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

  9. Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco. (United States)

    da Silva, Leonardo F; Dias, Cristiano V; Cidade, Luciana C; Mendes, Juliano S; Pirovani, Carlos P; Alvim, Fátima C; Pereira, Gonçalo A G; Aragão, Francisco J L; Cascardo, Júlio C M; Costa, Marcio G C


    Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).

  10. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

    DEFF Research Database (Denmark)

    Zargar, K.; Saville, R.; Phelan, R. M.;


    an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (Csd...... similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding...

  11. Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. (United States)

    Azcarate-Peril, M Andrea; Bruno-Bárcena, Jose M; Hassan, Hosni M; Klaenhammer, Todd R


    Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus. Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of Lactobacillus gasseri and Bifidobacterium lactis, none of the other strains harbored genes for oxalate utilization.

  12. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast. (United States)

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F


    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  13. Histidine decarboxylase deficiency causes Tourette syndrome: parallel findings in humans and mice (United States)

    Baldan, Lissandra Castellan; Rapanelli, Maximiliano; Crowley, Michael; Anderson, George M.; Loring, Erin; Gorczyca, Roxanne; Billingslea, Eileen; Wasylink, Suzanne; Panza, Kaitlyn E.; Ercan-Sencicek, A. Gulhan; Krusong, Kuakarun; Leventhal, Bennett L.; Ohtsu, Hiroshi; Bloch, Michael H.; Hughes, Zoë A.; Krystal, John H.; Mayes, Linda; de Araujo, Ivan; Ding, Yu-Shin; State, Matthew W.; Pittenger, Christopher


    Tourette syndrome (TS) is characterized by tics, sensorimotor gating deficiencies, and abnormalities of cortico-basal ganglia circuits. A mutation in histidine decarboxylase (Hdc), the key enzyme for the biosynthesis of histamine (HA), has been implicated as a rare genetic cause. Hdc knockout mice exhibited potentiated tic-like stereotypies, recapitulating core phenomenology of TS; these were mitigated by the dopamine D2 antagonist haloperidol, a proven pharmacotherapy, and by HA infusion into the brain. Prepulse inhibition was impaired in both mice and humans carrying Hdc mutations. HA infusion reduced striatal dopamine (DA) levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. Dopamine D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm HDC deficiency as a rare cause of TS and identify histamine-dopamine interactions in the basal ganglia as an important locus of pathology. PMID:24411733

  14. Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi. (United States)

    Carrillo, Carolina; Serra, María P; Pereira, Claudio A; Huber, Alejandra; González, Nélida S; Algranati, Israel D


    Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

  15. Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

    Institute of Scientific and Technical Information of China (English)

    Takahiro Nemoto; Shunichiro Kubota; Hideyuki Ishida; Nobuo Murata; Daijo Hashimoto


    AIM: To investigate the expressions of omithine decarboxylase (ODC), MMP-2, and Erk, and their relationship in human colon tumors.METHODS: ODC activity, MMP-2 expression, and mitogenactivated protein (MAP) kinase activity (Erk phosphorylation) were determined in 58 surgically removed human colon tumors and their adjacent normal tissues, using [1-14C]-ornithine as a substrate, ELISA assay, and Western blotting, respectively.RESULTS: ODC activity, MMP-2 expression, and Erk phosphorylation were significantly elevated in colon tumors, compared to those in adjacent normal tissues. A significant correlation was observed between ODC activities and MMP-2 levels.CONCLUSION: This is the first report showing a significant correlation between ODC activities and MMP-2 levels in human colon tumors. As MMP-2 is involved in cancer invasion and metastasis, and colon cancer overexpresses ODC, suppression of ODC expression may be a rational approach to treat colon cancer which overexpresses ODC.

  16. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase. (United States)

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan


    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  17. The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana. (United States)

    Yunus, Ian Sofian; Liu, Yu-Chi; Nakamura, Yuki


    In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

  18. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P. (TGRI); (Toronto)


    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  19. Aprūpes prioritātes C hepatīta saslimšanas gadījumā, pacientu skatījuma


    Freimane, Arina


    Bakalaura darba „Aprūpes prioritātes C hepatīta saslimšanas gadījumā, pacientu skatījumā” mērķis – noteikt aprūpes prioritātes pacientu skatījumā C hepatīta saslimšanas gadījumā. K.Rojas „Adaptācijas modelis” nodrošina māsas piemērotu pieeju pacientam ar C hepatītu. Tika veikta literatūras analīze. Tika veikts pētījums. Darba teorētiskajā daļā ir informācija par vīrusa C hepatītu, māsas darba funkciju pacietu aprūpē, stresu, cilvēka vajadzībām. Darba pētnieciskajā daļā tiek ...

  20. Fluorescent labeling of newborn dentate granule cells in GAD67-GFP transgenic mice: a genetic tool for the study of adult neurogenesis.

    Directory of Open Access Journals (Sweden)

    Shengli Zhao

    Full Text Available Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP(+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP(+ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.

  1. Komandas darbs ar ģimenēm bērna pamešanas novārtā gadījumos



    Diplomdarba tēma ir” Komandas darbs ar ģimenēm bērna pamešanas novārtā gadījumos”. Tā mērķis ir teorētiski analizēt, praktiski pētīt un pamatot komandas darbu ar ģimenēm bērna pamešanas novārtā gadījumos. Lai sasniegtu darba mērķi, tika izvirzīti uzdevumi: raksturot bērna pamešanu novārtā ģimenē, analizēt komandas darba pieeju sociālā darba praksē, analizēt bērna pamešanu novārtā Latvijas likumdošanas un normatīvo dokumentu skatījumā, pētīt dažādu institūciju speciālistu viedokli par koman...

  2. 广泛性焦虑障碍的治疗进展%The Recent Advances in the Treatment of Generalized Anxiety Disorder (GAD)

    Institute of Scientific and Technical Information of China (English)

    沈鑫华; 沈仲夏


    The treatment options of generalized anxiety disorder include psychological therapies and pharmacological treatments. The highly utilized type of psychotherapy is CBT. Antidepressants include Selective serotonin reuptake inhibitors ( SSRIs) and serotonin - norepinephrine reuptake inhibitors ( SNRIs) are considered first line treatment options, and Sertraline is recommended by NICE as the initial treatment for GAD. The second generation antip-sychotics such as Quetiapine monotherapy can be used in refractory GAD. If a particular drug is effective, the optimal treatment duration is generally one year.%广泛性焦虑障碍心理治疗、药物治疗均可选择.心理治疗主要是CBT.药物治疗主要是抗抑郁药物,SSRI、SNRI是一线选择,NICE推荐舍曲林作为初始治疗药物.对于难治性的患者,可以试用第二代抗精神病药物,如喹硫平.如果药物治疗有效,最佳治疗时间为一年.

  3. Molecular screening of wine lactic acid bacteria degrading hydroxycinnamic acids. (United States)

    de las Rivas, Blanca; Rodríguez, Héctor; Curiel, José Antonio; Landete, José María; Muñoz, Rosario


    The potential to produce volatile phenols from hydroxycinnamic acids was investigated for lactic acid bacteria (LAB) isolated from Spanish grape must and wine. A PCR assay was developed for the detection of LAB that potentially produce volatile phenols. Synthetic degenerate oligonucleotides for the specific detection of the pdc gene encoding a phenolic acid decarboxylase were designed. The pdc PCR assay amplifies a 321 bp DNA fragment from phenolic acid decarboxylase. The pdc PCR method was applied to 85 strains belonging to the 6 main wine LAB species. Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus pentosaceus strains produce a positive response in the pdc PCR assay, whereas Oenococcus oeni, Lactobacillus hilgardii, and Leuconostoc mesenteroides strains did not produce the expected PCR product. The production of vinyl and ethyl derivatives from hydroxycinnamic acids in culture media was determined by high-performance liquid chromatography. A relationship was found between pdc PCR amplification and volatile phenol production, so that the LAB strains that gave a positive pdc PCR response produce volatile phenols, whereas strains that did not produce a PCR amplicon did not produce volatile phenols. The proposed method could be useful for a preliminary identification of LAB strains able to produce volatile phenols in wine.

  4. Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation. (United States)

    van Beek, S; Priest, F G


    Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.

  5. Ornithine decarboxylase antizyme finder (OAF: Fast and reliable detection of antizymes with frameshifts in mRNAs

    Directory of Open Access Journals (Sweden)

    Atkins John F


    Full Text Available Abstract Background Ornithine decarboxylase antizymes are proteins which negatively regulate cellular polyamine levels via their affects on polyamine synthesis and cellular uptake. In virtually all organisms from yeast to mammals, antizymes are encoded by two partially overlapping open reading frames (ORFs. A +1 frameshift between frames is required for the synthesis of antizyme. Ribosomes change translation phase at the end of the first ORF in response to stimulatory signals embedded in mRNA. Since standard sequence analysis pipelines are currently unable to recognise sites of programmed ribosomal frameshifting, proper detection of full length antizyme coding sequences (CDS requires conscientious manual evaluation by a human expert. The rapid growth of sequence information demands less laborious and more cost efficient solutions for this problem. This manuscript describes a rapid and accurate computer tool for antizyme CDS detection that requires minimal human involvement. Results We have developed a computer tool, OAF (ODC antizyme finder for identifying antizyme encoding sequences in spliced or intronless nucleic acid sequenes. OAF utilizes a combination of profile hidden Markov models (HMM built separately for the products of each open reading frame constituting the entire antizyme coding sequence. Profile HMMs are based on a set of 218 manually assembled antizyme sequences. To distinguish between antizyme paralogs and orthologs from major phyla, antizyme sequences were clustered into twelve groups and specific combinations of profile HMMs were designed for each group. OAF has been tested on the current version of dbEST, where it identified over six thousand Expressed Sequence Tags (EST sequences encoding antizyme proteins (over two thousand antizyme CDS in these ESTs are non redundant. Conclusion OAF performs well on raw EST sequences and mRNA sequences derived from genomic annotations. OAF will be used for the future updates of the RECODE

  6. Propuesta de fortalecimiento de la gesti?n del gobierno aut?nomo descentralizado (GAD) del cant?n Mej?a a trav?s de mejoramiento de los servicios que ofrece el municipio a las empresas



    El objetivo primordial de un gobierno central o local es lograr el bienestar de la poblaci?n ubicada en su territorio. El Diagnostico de la situaci?n actual del Cant?n Mej?a, como caso de estudio de la presente investigaci?n, se realiz? a base de los diferentes documentos proporcionados por el GAD del cant?n Mej?a, SENPLADES, INEC y entrevistas realizadas a diferentes funcionarios de la municipalidad. La Propuesta de Fortalecimiento de la Gesti?n del GAD del cant?n Mej?a integra tres el...

  7. Role of the multidrug resistance regulator MarA in global regulation of the hdeAB acid resistance operon in Escherichia coli. (United States)

    Ruiz, Cristian; McMurry, Laura M; Levy, Stuart B


    MarA, a transcriptional regulator in Escherichia coli, affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of hdeAB and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of hdeAB by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of hdeAB. Moreover, repression of hdeAB by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of hdeAB by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on hdeAB under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of hdeAB expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of hdeAB by MarA depends on pH, growth phase, and other regulators of hdeAB and is associated with reduced resistance to acid conditions.

  8. Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase. (United States)

    Kellett, Whitney F; Brunk, Elizabeth; Desai, Bijoy J; Fedorov, Alexander A; Almo, Steven C; Gerlt, John A; Rothlisberger, Ursula; Richards, Nigel G J


    The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.

  9. FcWRKY70, a WRKY protein of Fortunella crassifolia, functions in drought tolerance and modulates putrescine synthesis by regulating arginine decarboxylase gene. (United States)

    Gong, Xiaoqing; Zhang, Jingyan; Hu, Jianbing; Wang, Wei; Wu, Hao; Zhang, Qinghua; Liu, Ji-Hong


    WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report functional characterization of a Group III WRKY gene (FcWRKY70) from Fortunella crassifolia. FcWRKY70 was greatly induced by drought and abscisic acid, but slightly or negligibly by salt and cold. Overexpression of FcWRKY70 in tobacco (Nicotiana nudicaulis) and lemon (Citrus lemon) conferred enhanced tolerance to dehydration and drought stresses. Transgenic tobacco and lemon exhibited higher expression levels of ADC (arginine decarboxylase), and accumulated larger amount of putrescine in comparison with wild type (WT). Treatment with D-arginine, an inhibitor of ADC, caused transgenic tobacco plants more sensitive to dehydration. Knock-down of FcWRKY70 in kumquat down-regulated ADC abundance and decreased putrescine level, accompanied by compromised dehydration tolerance. The promoter region of FcADC contained two W-box elements, which were shown to be interacted with FcWRKY70. Taken together, our data demonstrated that FcWRKY70 functions in drought tolerance by, at least partly, promoting production of putrescine via regulating ADC expression.

  10. Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide. (United States)

    Nagasaki, Toshihiro; Hongo, Yuki; Koito, Tomoko; Nakamura-Kusakabe, Ikumi; Shimamura, Shigeru; Takaki, Yoshihiro; Yoshida, Takao; Maruyama, Tadashi; Inoue, Koji


    It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

  11. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato. (United States)

    Pandey, Roopali; Gupta, Aarti; Chowdhary, Anuj; Pal, Ram Krishna; Rajam, Manchikatla Venkat


    Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches.

  12. Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli (United States)

    Kim, Junyoung; Seo, Hyung-Min; Bhatia, Shashi Kant; Song, Hun-Seok; Kim, Jung-Ho; Jeon, Jong-Min; Choi, Kwon-Young; Kim, Wooseong; Yoon, Jeong-Jun; Kim, Yun-Gon; Yang, Yung-Hun


    Itaconate, a C5 unsaturated dicarboxylic acid, is an important chemical building block that is used in manufacturing high-value products, such as latex and superabsorbent polymers. Itaconate is produced by fermentation of sugars by the filamentous fungus Aspergillus terreus. However, fermentation by A. terreus involves a long fermentation period and the formation of various byproducts, resulting in high production costs. E. coli has been developed as an alternative for producing itaconate. However, fermentation of glucose gives low conversion yields and low productivity. Here, we report the whole-cell bioconversion of citrate to itaconate with enhanced aconitase and cis-aconitate decarboxylase activities by controlling the expression of multiple cadA genes. In addition, this bioconversion system does not require the use of buffers, which reduces the production cost and the byproducts released during purification. Using this whole-cell bioconversion system, we were able to catalyze the conversion of 319.8 mM of itaconate (41.6 g/L) from 500 mM citrate without any buffer system or additional cofactors, with 64.0% conversion in 19 h and a productivity of 2.19 g/L/h. Our bioconversion system suggests very high productivity for itaconate production. PMID:28051098

  13. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation. (United States)

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem


    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

  14. Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

    Directory of Open Access Journals (Sweden)

    Atkins John F


    Full Text Available Abstract Background The antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine. Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC, an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3 is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis. Methods To see if mutations in the OAZ3 gene are responsible for some cases of male infertility, we sequenced and evaluated the genomic DNA of 192 infertile men, 48 men of known paternity, and 34 African aborigines from the Mbuti tribe in the Democratic Republic of the Congo. The coding sequence of OAZ3 was further screened for polymorphisms by SSCP analysis in the infertile group and an additional 250 general population controls. Identified polymorphisms in the OAZ3 gene were further subjected to a haplotype analysis using PHASE 2.02 and Arlequin 2.0 software programs. Results A total of 23 polymorphisms were identified in the promoter, exons or intronic regions of OAZ3. The majority of these fell within a region of less than two kilobases. Two of the polymorphisms, -239 A/G in the promoter and 4280 C/T, a missense polymorphism in exon 5, may show evidence of association with male infertility. Haplotype analysis identified 15 different haplotypes, which can be separated into two divergent clusters. Conclusion Mutations in the OAZ3 gene are not a common cause of male infertility. However, the presence of the two divergent haplotypes at high frequencies in all three of our subsamples (infertile, control, African suggests that they have been maintained in the genome by balancing selection, which was supported by a test of Tajima's D statistic. Evidence for natural selection in this region implies that these haplotypes

  15. GABA shunt and polyamine degradation pathway on γ-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia. (United States)

    Yang, Runqiang; Guo, Qianghui; Gu, Zhenxin


    GABA shunt and polyamine degradation pathway on γ-aminobutyric acid (GABA) accumulation in germinating fava bean under hypoxia was investigated. GABA content, GAD and DAO activity were significantly increased under hypoxia treatment. Glu and polyamine contents enhanced largely and thus supplied as sufficient substrates for GABA formation. In contrast, GABA content decreased, mainly in the embryo, after removing the hypoxia stress. DAO activity, Glu and polyamines contents decreased, while an increment of GAD activity was observed. This indicated that GAD activity can be not only regulated by hypoxia, but by the rapid growth of embryo after the recovery from hypoxia stress. When treated with AG, DAO activity was almost inhibited completely, and the GABA content decreased by 32.96% and 32.07% after treated for 3 and 5 days, respectively. Hence, it can be inferred that about 30% of GABA formed in germinating fava bean under hypoxia was supplied by polyamine degradation pathway.

  16. Varas un seksualitātes mijiedarbe: nepilngadīgo grūtnieču gadījums


    Mileiko, Ilze


    Darba nosaukums - Varas un seksualitātes mijiedarbe: nepilngadīgo grūtnieču gadījums. Darbā analizēts, kādos veidos diskursīvā vara saistīta ar seksualitātes politiku un skatīts, kā varas attiecības sabiedrībā ietekmē politiku, kas orientēta uz pusaudzēm grūtniecēm. Pētījuma metode – mediju rakstu, komentāru un normatīvo aktu diskursīva analīze. Secinājumi. Diskursu pusaudžu grūtniecību veido seši diskursīvie ietvari, kas katrs pozicionē to no atšķirīga skata punkta. Katra ietvara satu...

  17. Intravenous immunoglobulin in patients with anti-GAD antibody-associated neurological diseases and patients with inflammatory myopathies: effects on clinicopathological features and immunoregulatory genes. (United States)

    Dalakas, Marinos C


    Controlled trials with intravenous immunoglobulin (IVIg) were conducted in patients with Stiff-Person Syndrome (SPS) and dermatomyositis (DM), two humorally mediated neurological disorders, and in inclusion body myositis (IBM), a T-cell-mediated inflammatory myopathy. The clinical efficacy was compared with alterations on tissue expression of complement, cytokines, chemokines, adhesion molecules, and immunoregulatory genes. The following patients were randomized in three separate trials to receive IVIg or placebo for 3 mo: (a) 16 patients with anti-GAD antibody-positive SPS; (b) 15 patients with DM resistant to therapies; and (c) 19 patients with IBM. After a washout, they crossed to the alternative therapy for another 3 mo. Efficacy was based on the difference in the respective disease scores from baseline to the second and third month of the infusions. In patients with SPS and DM, the scores changed positively and significantly from months 1 through 3, but returned to baseline when the patients crossed to placebo. In contrast, the scores in the placebo-randomized group remained constant or worsened from months 1 to 3, but improved significantly after crossing to IVIg. The muscle scores of patients with IBM did not significantly change between IVIg or placebo. In SPS, the anti-GAD65 antibody titers declined after IVIg but not after placebo. In DM, there was reduction of complement consumption, interception of membranolytic attack complex formation, downregulation of inflammation, fibrosis, cytokines, chemokines and adhesion molecules, and alterations in thousands of immunoregulatory genes. We conclude that IVIg is a safe and effective therapy for patients with SPS and DM unresponsive to other agents. In tissues, IVIg restores tissue cytoarchitecture by suppressing the inflammatory mediators at the protein, mRNA, and gene level.

  18. Characterization of GABAergic neurons in rapid-eye-movement sleep controlling regions of the brainstem reticular formation in GAD67-green fluorescent protein knock-in mice. (United States)

    Brown, Ritchie E; McKenna, James T; Winston, Stuart; Basheer, Radhika; Yanagawa, Yuchio; Thakkar, Mahesh M; McCarley, Robert W


    Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro. Small (10-15 microm) or medium-sized (15-25 microm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3-12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.

  19. Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase. (United States)

    Harris, T K; Washabaugh, M W


    Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (phi C(2) = 0.98 +/- 0.06) and HETDP C(alpha)-L (phi C(alpha) = 1.01 +/- 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (phi = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] or = 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.

  20. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.


    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

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

    Energy Technology Data Exchange (ETDEWEB)

    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)


    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.

  2. Effects of Teucrium polium aerial parts extracts on malonyl-CoA decarboxylase level.

    Directory of Open Access Journals (Sweden)

    Durdi Qujeq


    Full Text Available Malonyl-CoA decarboxylase (MCD is an enzyme involved in the decarboxylation of malonyl-CoA to acetyl-CoA. In order to explore the hypothesis that the changing plant materials’ MCD activity level can serve as therapy to diabetics, the effect of Teucrium polium compounds was studied in a diabetic rat model. In this experimental study, two groups of rats, a control and a diabetic group, each including six rats, were used. At the end of the experiment, all rats were exterminated by ether anesthesia, their pancreases removed and dissected. Isolated rat pancreas was cultured in buffers with or without 100-500µg/l T. polium aerial parts extracts containing arginine and leucine. MCD and insulin levels were measured after culture at 37°C and 5% CO2, for 1, 3 and 5 days. Results showed that T. polium aqueous and the alcoholic extract decreased MCD activity. Present data also indicate that incubation of pancreatic tissue at a concentration of 2.8 and 16.7 mmol/L glucose stimulated insulin release. For the first time it seems that aqueous and alcoholic extracts of this plant decreased MCD activity.

  3. Simultaneous silencing of two arginine decarboxylase genes alters development in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Diana eSánchez-Rangel


    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.

  4. Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain. (United States)

    Bessho, Yuki; Iwakoshi-Ukena, Eiko; Tachibana, Tetsuya; Maejima, Sho; Taniuchi, Shusuke; Masuda, Keiko; Shikano, Kenshiro; Kondo, Kunihiro; Furumitsu, Megumi; Ukena, Kazuyoshi


    In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

  5. Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier. (United States)

    Peper, Stephanie; Kara, Selin; Long, Wei Sing; Liese, Andreas; Niemeyer, Bernd


    If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

  6. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex. (United States)

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J


    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  7. Ornithine decarboxylase expression in the small intestine of broilers submitted to feed restriction and glutamine supplementation

    Directory of Open Access Journals (Sweden)

    AV Fischer da Silva


    Full Text Available Six hundred and forty one-day-old Cobb male broilers were used to evaluate ornithine decarboxylase (ODC expression in the mucosa of the small intestine. Birds were submitted to early feed restriction from 7 to 14 days of age. The provided feed was supplemented with glutamine. A completely randomized design with a 2 x 2 factorial arrangement was used (with or without glutamine, with or without feed restriction. Restricted-fed birds were fed at 30% the amount of the ad libitum fed group from 7 to 14 days of age. Glutamine was added at the level of 1% in the diet supplied from 1 to 28 days of age. Protein concentration in the small intestine mucosa was determined, and ODC expression at 7, 14, 21, and 28 days of age was evaluated by dot blotting. ODC was present in the mucosa of broilers, and the presence of glutamine in the diet increased ODC activation. Glutamine prevented mucosa atrophy by stimulating protein synthesis, and was effective against the effects of feed restriction. Dot blotting can be used to quantify ODC expression in the intestinal mucosa of broilers.

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

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


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

  9. The role of arginine decarboxylase in modulating the sensitivity of barley to ozone. (United States)

    Rowland-Bamford, A J; Borland, A M; Lea, P J; Mansfield, T A


    Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.

  10. Highly active and stable oxaloacetate decarboxylase Na⁺ pump complex for structural analysis. (United States)

    Inoue, Michio; Li, Xiaodan


    The oxaloacetate decarboxylase primary Na(+) pump (Oad) produces energy for the surviving of some pathogenic bacteria under anaerobic conditions. Oad composes of three subunits: Oad-α, a biotinylated soluble subunit and catalyzes the decarboxylation of oxaloacetate; Oad-β, a transmembrane subunit and functions as a Na(+) pump; and Oad-γ, a single transmembrane α-helical anchor subunit and assembles Oad-α/β/γ complex. The molecular mechanism of Oad complex coupling the exothermic decarboxylation to generate the Na(+) electrochemical gradient remains unsolved. Our biophysical and biochemical studies suggested that the stoichiometry of Oad complex from Vibrio cholerae composed of α, β, γ in 4:2:2 stoichiometry not that of 4:4:4. The high-resolution structure determination of the Oad complex would reveal the energetic transformation mechanism from the catalytical soluble α subunit to membrane β subunit. Sufficient amount stable, conformational homogenous and active Oad complex with the right stoichiometry is the prerequisite for structural analysis. Here we report an easy and reproducible protocol to obtain high quantity and quality Oad complex protein for structural analysis.

  11. Role of the Sulfonium Center in Determining the Ligand Specificity of Human S-Adenosylmethionine Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Bale, Shridhar; Brooks, Wesley; Hanes, Jeremiah W.; Mahesan, Arnold M.; Guida, Wayne C.; Ealick, Steven E.; (Moffitt); (Cornell)


    S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requirement for a positive charge at the position equivalent to the sulfonium of S-adenosylmethionine. We investigated the ligand specificity of AdoMetDC through crystallography, quantum chemical calculations, and stopped-flow experiments. We determined crystal structures of the enzyme cocrystallized with 5{prime}-deoxy-5{prime}-dimethylthioadenosine and 5{prime}-deoxy-5{prime}-(N-dimethyl)amino-8-methyladenosine. The crystal structures revealed a favorable cation-{pi} interaction between the ligand and the aromatic side chains of Phe7 and Phe223. The estimated stabilization from this interaction is 4.5 kcal/mol as determined by quantum chemical calculations. Stopped-flow kinetic experiments showed that the rate of the substrate binding to the enzyme greatly depends on Phe7 and Phe223, thus supporting the importance of the cation-{pi} interaction.

  12. Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1. (United States)

    Zou, Yu; Hu, Wenzhong; Jiang, Aili; Tian, Mixia


    Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

  13. Inhibition of scratching behaviour caused by contact dermatitis in histidine decarboxylase gene knockout mice. (United States)

    Seike, M; Ikeda, M; Kodama, H; Terui, T; Ohtsu, H


    A neuronal system dedicated to itch consists of primary afferent and spinothalamic projection neurons. Histamine is thought to be one of the main mediators for the transmission of itch sensation. However, there are little available information on the role of histamine in scratching behaviour and sensory transmission of atopic dermatitis and chronic eczema. In the present study, the role of histamine in scratching behaviour and neural conduction of sensation in the chronic eczema model was investigated by using l-histidine decarboxylase (HDC) gene knockout mice lacking histamine. The chronic contact dermatitis was induced with daily application of diphenylcyclopropenone (DCP) on a hind paw of HDC (+/+) and HDC (-/-) mice for 2 months. The observation of scratching behaviour and the hot-plate test were performed in both mice. Histological studies were performed in the skin and spinal cord tissues. Histological examination revealed that both HDC (+/+) and HDC (-/-) mice displayed the similar extent of inflammatory cell infiltration, hyperplastic epidermis and newly spreading of neuronal processes in the skin tissue. Scratching behaviour was exclusively induced in HDC (+/+) mice, whereas it was barely observed in HDC (-/-) mice. The expression of c-Fos was specifically upregulated in HDC (+/+) mice in lamina I of the spinal dorsal horn following repeated DCP application. Scratching behaviour in chronic contact dermatitis in mice was thought mainly mediated with histamine. The afferent pathway of sensation in chronic contact dermatitis model may connect with the central nervous system through lamina I of the spinal dorsal horn.

  14. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG


    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  15. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C


    Uroporphyrinogen decarboxylase (URO-D; EC, the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  16. Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma. (United States)

    Lozier, Ann M; Rich, Maria E; Grawe, Anissa Pedersen; Peck, Anderson S; Zhao, Ping; Chang, Anthony Ting-Tung; Bond, Jeffrey P; Sholler, Giselle Saulnier


    LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

  17. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear. (United States)

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher


    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.

  18. Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme. (United States)

    Winer, L; Vinkler, C; Apelbaum, A


    A partially purified preparation of arginine decarboxylase (EC, a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.

  19. Lactic acid bacterial cell factories for gamma-aminobutyric acid. (United States)

    Li, Haixing; Cao, Yusheng


    Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.

  20. The gamma-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp PCC 6803

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, W; Brune, D; Vermaas, WFJ


    A traditional 2-oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2-oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Delta sll1981, Delta slr0370, Delta slr1022 and combinations thereof, deficient in 2-oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in gamma-aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N-acetylornithine aminotransferase, encoded by slr1022, was shown to also function as gamma-aminobutyrate aminotransferase, catalysing gamma-aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact gamma-aminobutyrate shunt is present in Synechocystis. The Delta sll1981 strain, lacking 2-oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Delta slr1022 and Delta slr0370 strains and the Delta sll1981/Delta slr1022 and Delta sll1981/Delta slr0370 double mutants was reduced to 20-40% of that in wild type, suggesting that the gamma-aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2-oxoglutarate decarboxylase. C-13-stable isotope analysis indicated that the gamma-aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2-oxoglutarate decarboxylase bypass, the gamma-aminobutyrate shunt is a major contributor to flux from 2-oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.

  1. Vulnerability of hippocampal GABA-ergic interneurons to kainate-induced excitotoxic injury during old age. (United States)

    Shetty, Ashok K; Hattiangady, Bharathi; Rao, Muddanna S


    Hippocampal inhibitory interneurons expressing glutamate decarboxylase-67 (GAD-67) considerably decline in number during old age. Studies in young adult animals further suggest that hippocampal GAD-67+ interneuron population is highly vulnerable to excitotoxic injury. However, the relative susceptibility of residual GAD-67+ interneurons in the aged hippocampus to excitotoxic injury is unknown. To elucidate this, using both adult and aged F344 rats, we performed stereological counting of GAD-67+ interneurons in different layers of the dentate gyrus and CA1 & CA3 sub-fields, at 3 months post-excitotoxic hippocampal injury inflicted through an intracerebroventricular administration of kainic acid (KA). Substantial reductions of GAD-67+ interneurons were found in all hippocampal layers and sub-fields after KA-induced injury in adult animals. Contrastingly, there was no significant change in GAD-67+ interneuron population in any of the hippocampal layers and sub-fields following similar injury in aged animals. Furthermore, the stability of GAD-67+ interneurons in aged rats after KA was not attributable to milder injury, as the overall extent of KA-induced hippocampal principal neuron loss was comparable between adult and aged rats. Interestingly, because of the age-related disparity in vulnerability of interneurons to injury, the surviving GAD-67+ interneuron population in the injured aged hippocampus remained comparable to that observed in the injured adult hippocampus despite enduring significant reductions in interneuron number with aging. Thus, unlike in the adult hippocampus, an excitotoxic injury to the aged hippocampus does not result in significantly decreased numbers of GAD-67+ interneurons. Persistence of GAD-67+ interneuron population in the injured aged hippocampus likely reflects an age-related change in the response of GAD-67+ interneurons to excitotoxic hippocampal injury. These results have implications towards understanding mechanisms underlying the

  2. Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein. (United States)

    Zeng, X; Farrenkopf, B; Hohmann, S; Dyda, F; Furey, W; Jordan, F


    Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants. (United States)

    Wi, Soo Jin; Kim, Woo Taek; Park, Ky Young


    Polyamines (PAs), such as putrescine, spermidine, and spermine, are present in all living organism and implicate in a wide range of cellular physiological processes. We have used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Sense construct of full-length cDNA for S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in PA biosynthesis, from carnation (Dianthus caryophyllus L.) flower was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. Several transgenic lines overexpressing SAMDC gene under the control of cauliflower mosaic virus 35S promoter accumulated soluble total PAs by 2.2 (S16-S-4) to 3.1 (S16-S-1) times than wild-type plants. The transgenic tobacco did not show any difference in organ phenotype compared to the wild-type. The number and weight of seeds increased, and net photosynthetic rate also increased in transgenic plants. Stress-induced damage was attenuated in these transgenic plants, in the symptom of visible yellowing and chlorophyll degradation after all experienced stresses such as salt stress, cold stress, acidic stress, and abscisic acid treatment. H2O2-induced damage was attenuated by spermidine treatment. Transcripts for antioxidant enzymes (ascorbate peroxidase, manganase superoxide dismutase, and glutathione S-transferase) in transgenic plants and GUS activity transformed with SAMDC promoter::GUS fusion were induced more significantly by stress treatment, compared to control. These results that the transgenic plants with sense SAMDC cDNA are more tolerant to abiotic stresses than wild-type plants suggest that PAs may play an important role in contributing stress tolerance in plants.

  4. Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium. (United States)

    Vaccaro, J A; Crane, E J; Harris, T K; Washabaugh, M W


    Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine. (United States)

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W


    Brewers' yeast pyruvate decarboxylase (EC, a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Meat consumption, ornithine decarboxylase gene polymorphism, and outcomes after colorectal cancer diagnosis

    Directory of Open Access Journals (Sweden)

    Jason A Zell


    Full Text Available Background: Dietary arginine and meat consumption are implicated in colorectal cancer (CRC progression via polyamine-dependent processes. Polymorphism in the polyamine-regulatory gene, ornithine decarboxylase 1 (Odc1, rs2302615 is prognostic for CRC-specific mortality. Here, we examined joint effects of meat consumption and Odc1 polymorphism on CRC-specific mortality. Materials and Methods: The analytic cohort was comprised of 329 incident stage I-III CRC cases diagnosed 1994-1996 with follow- up through March 2008. Odc1 genotyping was conducted using primers that amplify a 172-bp fragment containing the polymorphic base at +316. Dietary questionnaires were administered at cohort entry. Multivariate Cox proportional hazards regression analysis for CRC-specific mortality was stratified by tumor, node, metastasis (TNM stage, and adjusted for clinically relevant variables, plus meat consumption (as a continuous variable, i.e., the number of medium-sized servings/week, Odc1 genotype, and a term representing the meat consumption and Odc1 genotype interaction. The primary outcome was the interaction of Odc1 and meat intake on CRC-specific mortality, as assessed by departures from multiplicative joint effects. Results: Odc1 genotype distribution was 51% GG, 49% GA/AA. In the multivariate model, there was a significant interaction between meat consumption and Odc1 genotype, P-int = 0.01. Among Odc1 GA/AA CRC cases in meat consumption Quartiles 1-3, increased mortality risk was observed when compared to GG cases (adjusted hazards ratio (HR = 7.06 [95% CI 2.34-21.28] - a difference not found among cases in the highest dietary meat consumption Quartile 4. Conclusions: Effects of meat consumption on CRC-specific mortality risk differ based on genetic polymorphism at Odc1. These results provide further evidence that polyamine metabolism and its modulation by dietary factors such as meat may have relevance to CRC outcomes.

  7. Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius. (United States)

    Van Zyl, L J; Taylor, M P; Eley, K; Tuffin, M; Cowan, D A


    This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

  8. Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

    Directory of Open Access Journals (Sweden)

    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.

  9. Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development. (United States)

    Peters, Jennifer L; DeMars, Paul L; Collins, Lindsay M; Stoner, Julie A; Matsumoto, Hiroyuki; Komori, Naoka; Singh, Anil; Feasley, Christa L; Haddock, James A; Levine, Martin


    Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may

  10. [Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis]. (United States)

    Logvinovich, O S; Aksenova, G E


    Ornithine decarboxylase (ODC, EC 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.

  11. A tyrosine decarboxylase catalyzes the initial reaction of the salidroside biosynthesis pathway in Rhodiola sachalinensis. (United States)

    Zhang, Ji-Xing; Ma, Lan-Qing; Yu, Han-Song; Zhang, Hong; Wang, Hao-Tian; Qin, Yun-Fei; Shi, Guang-Lu; Wang, You-Nian


    Salidroside, the 8-O-β-D-glucoside of tyrosol, is the main bioactive component of Rhodiola species and is found mainly in the plant roots. It is well known that glucosylation of tyrosol is the final step in the biosynthesis of salidroside; however, the biosynthetic pathway of tyrosol and its regulation are less well understood. A summary of the results of related studies revealed that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. In this study, a cDNA clone encoding tyrosine decarboxylase (TyrDC) was isolated from Rhodiola sachalinensis A. Bor using rapid amplification of cDNA ends. The resulting cDNA was designated RsTyrDC. RNA gel-blot analysis revealed that the predominant sites of expression in plants are the roots and high levels of transcripts are also found in callus tissue culture. Functional analysis revealed that tyrosine was best substrate of recombinant RsTyrDC. The over-expression of the sense-RsTyrDC resulted in a marked increase of tyrosol and salidroside content, but the levels of tyrosol and salidroside were 274 and 412%, respectively, lower in the antisense-RsTyrDC transformed lines than those in the controls. The data presented here provide in vitro and in vivo evidence that the RsTyrDC can regulate the tyrosol and salidroside biosynthesis, and the RsTyrDC is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. sachalinensis.

  12. [Catabolism of carnitine: products of carnitine decarboxylase and carnitine dehydrogenase in vivo]. (United States)

    Seim, H; Löster, H; Strack, E


    1) Rats and mice were given large oral or subcutaneous doses of (-)-L-, (+)-D- and DL-carnitine (5 mg/g body weight). The carnitine metabolites, beta-methylcholine and acetonyltrimethylammonium, were isolated from the urine by special methods, and determined as their characteristic derivatives (2,4-dinitrophenylhydrazone and butyric ester) by thin-layer chromatography or photometry. 2) beta-Methylcholine, the product of carnitine decarboxylase, was not excreted, even when animals were heavily dosed with both carnitine isomers, with or without starvation. 3) After the administration of (+)-D- and DL-carnitine, both species excreted acetonyltrimethylammonium, which is already known as the spontaneous decarboxylation product of dehydrocarnitine (product of carnitine dehydrogenase) in bacteria. Injection of 0.71 mmol (+)-D-carnitine resulted in the excretion of 5.0 mumol (average) acetonyltrimethylammonium per mouse during the 48 h post injection. Under the same conditions, rats produced up to 40 mumol acetonyltrimethylammonium. The ratio of excreted acetonyltrimethylammonium to injected (+)-D-carnitine depended on the method of administration and the dose. 4) Production of the pharmacologically active (+)-acetyl-L-beta-methylcholine is not to be expected, following high exogenous doses of (-)-L-carnitine or (-)-acetyl-L-carnitine. The chief metabolites are trimethylamine, trimethylamine oxide and gamma-butyrobetaine (this journal 361, 1059), and both the (-)-L-carnitine pool and exogenous (-)-L-carnitine are dehydrogenated or decarboxylated only to a very small extent, if at all. When DL-carnitine is used therapeutically, the formation of acetonyltrimethylammonium must be taken into account.

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

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    Masome Zeinali


    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.

  14. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen (United States)

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


    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 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.

  15. A case of childhood stiff-person syndrome with striatal lesions: a possible entity distinct from the classical adult form. (United States)

    Sanefuji, Masafumi; Torisu, Hiroyuki; Kira, Ryutaro; Yamashita, Hiroshi; Ejima, Kazuna; Shigeto, Hiroshi; Takada, Yui; Yoshida, Keiko; Hara, Toshiro


    Parainfectious or autoimmune striatal lesions have been repeatedly described in children. We report a 7-year-old girl with painful muscle spasms, leading to the diagnosis of childhood stiff-person syndrome (SPS). Striatal lesions were demonstrated by diffusion-weighted magnetic resonance imaging (MRI) and single-photon emission computed tomography but not by conventional MRI. Autoantibodies against glutamic acid decarboxylase (GAD) were absent. Steroid pulse therapy and high-dose intravenous immunoglobulin resolved all the symptoms with slight sequelae. Childhood SPS may be characterized by absent anti-GAD antibodies and a transient benign clinical course, and it may have a pathomechanism distinct from that in adult SPS.

  16. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity. (United States)

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M; Park, Jin-Byung

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  17. Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis. (United States)

    Kim, Bokyung; Kim, Gyusik; Fujioka, Shozo; Takatsuto, Suguru; Choe, Sunghwa


    Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

  18. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. (United States)

    Desai, Bijoy J; Goto, Yuki; Cembran, Alessandro; Fedorov, Alexander A; Almo, Steven C; Gao, Jiali; Suga, Hiroaki; Gerlt, John A


    Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

  19. 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.

  20. Iekšējā komunikācija pašvaldībā: Dobeles novada gadījums (2013)


    Košinska, Egija


    Bakalaura darba tēma ir „Iekšējā komunikācija pašvaldībā: Dobeles novada gadījums (2013)”. Darbā ir izvirzīta problēma, vai lielisku sabiedrisko attiecību kritēriji un ideāli tipisko SA modeļu normatīvs pielietojums, kā arī komunikācijas kanālu un instrumentu daudzveidība kalpo kā orientieris Dobeles novada pašvaldības iekšējās komunikācijas procesos. Bakalaura darbā teorētiski apskatīta: iekšējā komunikācija, lieliskums sabiedriskajās attiecībās, pašvaldība un organizācija. Pētījum...

  1. Mutācijas riska gēnos pacientiem ar atkārtotiem melanomas saslimšanas gadījumiem ģimenē


    Veinalde, Rūta


    Melanoma ir bīstamākais ādas audzēja veids, kas attīstās pigmentu producējošo šūnu, melanocītu, nekontrolētas dalīšanās rezultātā. Melanomas kā multifaktoriālas saslimšanas attīstībā nozīme ir gan apkārtējās vides, gan ģenētisku riska faktoru mijiedarbībai. Galvenie līdz šim identificētie augsta riska melanomas jutības gēni ir CDKN2A, kurā mutācijas sastop apmēram pusē „melanomas ģimeņu” un CDK4, kurā mutācijas sastopamas mazāk kā 1% gadījumu. Latvijas populācijā līdz šim atrasta mutācija p.R...

  2. Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth. (United States)

    Kanjanaphachoat, Parawee; Wei, Bi-Yin; Lo, Shuen-Fang; Wang, I-Wen; Wang, Chang-Sheng; Yu, Su-May; Yen, Ming-Liang; Chiu, Sheng-Hsien; Lai, Chien-Chen; Chen, Liang-Jwu


    A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

  3. Biogenic Amine Degradation by Bacillus Species Isolated from Traditional Fermented Soybean Food and Detection of Decarboxylase-Related Genes. (United States)

    Eom, Jeong Seon; Seo, Bo Young; Choi, Hye Sun


    Biogenic amines in some food products present considerable toxicological risks as potential human carcinogens when consumed in excess concentrations. In this study, we investigated the degradation of the biogenic amines histamine and tyramine and the presence of genes encoding histidine and tyrosine decarboxylases and amine oxidase in Bacillus species isolated from fermented soybean food. No expression of histidine and tyrosine decarboxylase genes (hdc and tydc) were detected in the Bacillus species isolated (B. subtilis HJ0-6, B. subtilis D'J53-4, and B. idriensis RD13-10), although substantial levels of amine oxidase gene (yobN) expression were observed. We also found that the three selected strains, as non-biogenic amineproducing bacteria, were significantly able to degrade the biogenic amines histamine and tyramine. These results indicated that the selected Bacillus species could be used as a starter culture for the control of biogenic amine accumulation and degradation in food. Our study findings also provided the basis for the development of potential biological control agents against these biogenic amines for use in the food preservation and food safety sectors.

  4. Generating knock-in parasites: integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani. (United States)

    Roberts, Sigrid C; Kline, Chelsey; Liu, Wei; Ullman, Buddy


    Leishmania null mutants created by targeted gene replacement are typically complemented with chimeric episomes harboring the replaced gene in order to validate that the observed phenotype is due to the specific gene deletion. However, the current inventory of available episomes for complementation of genetic lesions in Leishmania is unstable in the absence of drug selection, and levels of gene expression cannot be controlled, especially in vivo. To circumvent this impediment, a strategy to re-introduce the targeted gene into the original chromosomal locus to generate "knock-in" parasites within selectable null backgrounds has been developed. A genomic fragment encompassing the ornithine decarboxylase locus and lacking heterologous DNA sequences was transfected into ornithine decarboxylase-deficient Leishmania donovani. The construct randomly integrated into either chromosomal allele by homologous recombination restoring polyamine prototrophy and revealing that LdODC was functionally expressed in the knock-in clones. This strategy offers a mechanism for complementing a genetic lesion amenable to positive selection in a manner that facilitates stable gene expression from its original locus in the absence of continuous drug pressure.

  5. Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine. (United States)

    Vuosku, Jaana; Jokela, Anne; Läärä, Esa; Sääskilahti, Mira; Muilu, Riina; Sutela, Suvi; Altabella, Teresa; Sarjala, Tytti; Häggman, Hely


    In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

  6. Adaptation and initial validation of the Patient Health Questionnaire - 9 (PHQ-9) and the Generalized Anxiety Disorder - 7 Questionnaire (GAD-7) in an Arabic speaking Lebanese psychiatric outpatient sample. (United States)

    Sawaya, Helen; Atoui, Mia; Hamadeh, Aya; Zeinoun, Pia; Nahas, Ziad


    The Patient Health Questionnaire - 9 (PHQ-9) and Generalized Anxiety Disorder - 7 (GAD-7) are short screening measures used in medical and community settings to assess depression and anxiety severity. The aim of this study is to translate the screening tools into Arabic and evaluate their psychometric properties in an Arabic-speaking Lebanese psychiatric outpatient sample. The patients completed the questionnaires, among others, prior to being evaluated by a clinical psychiatrist or psychologist. The scales' internal consistency and factor structure were measured and convergent and discriminant validity were established by comparing the scores with clinical diagnoses and the Psychiatric Diagnostic Screening Questionnaire - MDD subset (PDSQ - MDD). Results showed that the PHQ-9 and GAD-7 are reliable screening tools for depression and anxiety and their factor structures replicated those reported in the literature. Sensitivity and specificity analyses showed that the PHQ-9 is sensitive but not specific at capturing depressive symptoms when compared to clinician diagnoses whereas the GAD-7 was neither sensitive nor specific at capturing anxiety symptoms. The implications of these findings are discussed in reference to the scales themselves and the cultural specificity of the Lebanese population.

  7. Prevalência dos marcadores imunológicos Anti-GAD e Anti-IA2 em parentes de primeiro grau de diabéticos do tipo 1 em amostra da população da Grande São Paulo Prevalence of immunological markers (Anti-GAD and Anti-IA2 in first-degree relatives of patients with type 1 diabetes in Great São Paulo City

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    Paulo Roberto Cesarini


    Full Text Available O diabetes tipo 1A (DM1 é causado por mecanismo auto-imune contra células beta em indivíduos com predisposição genética. Auto-anticorpos anti-GAD e anti-IA2 são considerados importantes marcadores destas alterações, cuja prevalência variam, segundo a população estudada e história familiar. Dados sobre freqüência desses marcadores na população brasileira são escassos. OBJETIVOS: Avaliar a freqüência de anti-GAD e anti-IA2 em parentes de primeiro grau de portadores de DM1 (PDM1 em amostra da população da Grande São Paulo. MÉTODOS: Quarenta e oito jovens PDM1 foram recrutados junto a 36 propósitos assistidos em ambulatórios especializados em diabetes da Grande São Paulo, apresentando mediana de idade de 14,5 anos (6,7 a 17,9 anos. Os valores de referência do anti-GAD foram obtidos utilizado-se soros de 194 voluntários normais, sem antecedentes familiares de DM1, com idade de 9,7 a 64,0 anos (mediana de 13,4. Soros de 71 indivíduos normais com idade variando de 11,1 a 15,2 anos (Mi= 12,6 foram submetidos à dosagem de anti-IA2. As dosagens dos marcadores foram através do radioensaio (KRONUS®, Idaho, USA. Valores acima do 99º percentil obtido no grupo controle foram considerados positivos. RESULTADOS: O 99º percentil correspondeu ao valor 1,72 U/ml para o anti-GAD e 0,97 U/ml para o anti-IA2. Cinco indivíduos dos PDM1 (10,4% foram positivos para o anti-GAD, contra 0,5% dos controles (PBACKGROUND: Increasingly accurate prediction of Type1 Diabetes Mellitus (DM1, based on analysis of autoantibody markers, has become possible in first-degree relatives of patients with diabetes (PDM1. These markers indicate autoimmune process against pancreatic islet beta-cells. Anti-GAD and anti-IA2 are considered predictive of DM1, whose prevalences are considerably variable in different populations studied. There are few data about the frequency of these markers on the Brazilian population. The aim of this study is determine the

  8. 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


    The competitive S-adenosylmethionine decarboxylase (SAMdc; EC 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 effe

  9. Kv3.1b and Kv3.3 channel subunit expression in murine spinal dorsal horn GABAergic interneurones


    Nowak, A; Mathieson, H.R.; Chapman, R.J.; Janzsó, G.; Yanagawa, Y; Obata, K.; Szabo, G.; King, A. E.


    GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profil...

  10. Gene expression of ornithine decarboxylase, cyclooxygenase-2, and gastrin in atrophic gastric mucosa infected with Helicobacter pylori before and after eradication therapy. (United States)

    Konturek, Peter C; Rembiasz, Kazimierz; Konturek, Stanislaw J; Stachura, Jerzy; Bielanski, Wladyslaw; Galuschka, K; Karcz, Danuta; Hahn, Eckhart G


    H. pylori (Hp) -induced atrophic gastritis is a well-known risk factor for the development of gastric cancer. Whether Hp eradication can prevent or retard the progress of atrophy and metaplasia has been the topic of numerous studies but the subject remains controversial. Recently, the increased expression of ornithine decarboxylase (ODC), gastrin and cyclooxygenase (COX)-2 has been shown to be increased in premalignant lesions in gastric mucosa and to play an essential role in the malignant transformation. The aim of the study is to assess the effect of eradication therapy on atrophic gastritis and analyze the gene expression for ODC, COX-2 and gastrin in gastric mucosa after succesful eradication in patients with atrophic gastritis. Twenty patients with chronic atrophic gastritis including both corpus and antrum of the stomach were included in this study. Four antral mucosal biopsy specimens were obtained from antrum and four from corpus. The histopathologic evaluation of gastritis was based on Sydney classification of gastritis. All patients were Hp positive based on the [13C] urea breath test (UBT) and the presence of anti-Hp IgG and anti-CagA-antibodies detected by ELISA. The patients were then eradicated with triple therapy consiting of omeprazol (2 x 20 mg), amoxycillin (2 x 1 g) and clarithromycin (2 x 500 mg) for seven days and vitamin C 1 g/day for three months. In gastric mucosal samples obtained from the antrum and corpus before and after eradication, the mRNA expression for ODC, COX-2, and gastrin was assessed by reverse-transcription polymerase chain reaction (RT-PCR). In all patients the gastric secretory analysis was performed by measuring gastric acid output and serum gastrin levels. After triple therapy the successful eradication assessed by UBT was observed in 95% of patients. In 45% of patients the infection with CagA-positive Hp strain was observed. Three months after eradication a significant reduction in the gastric activity (neutrophilic

  11. CD226 rs763361 Is Associated with the Susceptibility to Type 1 Diabetes and Greater Frequency of GAD65 Autoantibody in a Brazilian Cohort (United States)

    Mattana, Teresa Cristina Colvara; Santos, Aritania Sousa; Fukui, Rosa Tsuneshiro; Mainardi-Novo, Debora Teixeira Oliveira; Costa, Vinícius Silva; Santos, Rosa Ferreira; Matioli, Sergio Russo; Rossi da Silva, Maria Elizabeth


    CD226 rs763361 variant increases susceptibility to type 1 diabetes (T1D) in Caucasians. There is no data about CD226 variants in the very heterogeneous Brazilian population bearing a wide degree of admixture. We investigated its association with T1D susceptibility, clinical phenotypes, and autoimmune manifestations (islet and extrapancreatic autoantibodies). Casuistry. 532 T1D patients and 594 controls in a case-control study. Initially, CD226 coding regions and boundaries were sequenced in a subset of 106 T1D patients and 102 controls. In a second step, two CD226 variants, rs763361 (exon 7) and rs727088 (3′ UTR region), involved with CD226 regulation, were genotyped in the entire cohort. C-peptide and autoantibody levels were determined. No new polymorphic variant was found. The variants rs763361 and rs727088 were in strong linkage disequilibrium. The TT genotype of rs763361 was associated with TID risk (OR = 1.503;  95%  CI = 1.135–1.991; P = 0.0044), mainly in females (P = 0.0012), greater frequency of anti-GAD autoantibody (31.9% × 24.5%; OR = 1.57; CI = 1.136–2.194; P = 0.0081), and lower C-peptide levels when compared to those with TC + CC genotypes (0.41 ± 0.30 ng/dL versus 0.70 ± 0.53 ng/dL P = 0.0218). Conclusions. The rs763361 variant of CD226 gene (TT genotype) was associated with susceptibility to T1D and with the degree of aggressiveness of the disease in T1D patients from Brazil. Ancestry had no effect. PMID:24891767

  12. CD226 rs763361 Is Associated with the Susceptibility to Type 1 Diabetes and Greater Frequency of GAD65 Autoantibody in a Brazilian Cohort

    Directory of Open Access Journals (Sweden)

    Teresa Cristina Colvara Mattana


    Full Text Available CD226 rs763361 variant increases susceptibility to type 1 diabetes (T1D in Caucasians. There is no data about CD226 variants in the very heterogeneous Brazilian population bearing a wide degree of admixture. We investigated its association with T1D susceptibility, clinical phenotypes, and autoimmune manifestations (islet and extrapancreatic autoantibodies. Casuistry. 532 T1D patients and 594 controls in a case-control study. Initially, CD226 coding regions and boundaries were sequenced in a subset of 106 T1D patients and 102 controls. In a second step, two CD226 variants, rs763361 (exon 7 and rs727088 (3′ UTR region, involved with CD226 regulation, were genotyped in the entire cohort. C-peptide and autoantibody levels were determined. No new polymorphic variant was found. The variants rs763361 and rs727088 were in strong linkage disequilibrium. The TT genotype of rs763361 was associated with TID risk (OR=1.503;  95%  CI=1.135–1.991; P=0.0044, mainly in females P=0.0012, greater frequency of anti-GAD autoantibody (31.9% × 24.5%; OR=1.57; CI=1.136–2.194; P=0.0081, and lower C-peptide levels when compared to those with TC + CC genotypes (0.41±0.30 ng/dL versus 0.70±0.53 ng/dL P=0.0218. Conclusions. The rs763361 variant of CD226 gene (TT genotype was associated with susceptibility to T1D and with the degree of aggressiveness of the disease in T1D patients from Brazil. Ancestry had no effect.

  13. The Roles of Organic Acids in C4 Photosynthesis. (United States)

    Ludwig, Martha


    Organic acids are involved in numerous metabolic pathways in all plants. The finding that some plants, known as C4 plants, have four-carbon dicarboxylic acids as the first product of carbon fixation showed these organic acids play essential roles as photosynthetic intermediates. Oxaloacetate (OAA), malate, and aspartate (Asp) are substrates for the C4 acid cycle that underpins the CO2 concentrating mechanism of C4 photosynthesis. In this cycle, OAA is the immediate, short-lived, product of the initial CO2 fixation step in C4 leaf mesophyll cells. The malate and Asp, resulting from the rapid conversion of OAA, are the organic acids delivered to the sites of carbon reduction in the bundle-sheath cells of the leaf, where they are decarboxylated, with the released CO2 used to make carbohydrates. The three-carbon organic acids resulting from the decarboxylation reactions are returned to the mesophyll cells where they are used to regenerate the CO2 acceptor pool. NADP-malic enzyme-type, NAD-malic enzyme-type, and phosphoenolpyruvate carboxykinase-type C4 plants were identified, based on the most abundant decarboxylating enzyme in the leaf tissue. The genes encoding these C4 pathway-associated decarboxylases were co-opted from ancestral C3 plant genes during the evolution of C4 photosynthesis. Malate was recognized as the major organic acid transferred in NADP-malic enzyme-type C4 species, while Asp fills this role in NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type plants. However, accumulating evidence indicates that many C4 plants use a combination of organic acids and decarboxylases during CO2 fixation, and the C4-type categories are not rigid. The ability to transfer multiple organic acid species and utilize different decarboxylases has been suggested to give C4 plants advantages in changing and stressful environments, as well as during development, by facilitating the balance of energy between the two cell types involved in the C4 pathway of CO2

  14. Inhibition of parvalbumin-expressing interneurons results in complex behavioral changes. (United States)

    Brown, J A; Ramikie, T S; Schmidt, M J; Báldi, R; Garbett, K; Everheart, M G; Warren, L E; Gellért, L; Horváth, S; Patel, S; Mirnics, Károly


    Reduced expression of the Gad1 gene-encoded 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of schizophrenia. GAD67 downregulation occurs in multiple interneuronal sub-populations, including the parvalbumin-positive (PVALB+) cells. To investigate the role of the PV-positive GABAergic interneurons in behavioral and molecular processes, we knocked down the Gad1 transcript using a microRNA engineered to target specifically Gad1 mRNA under the control of Pvalb bacterial artificial chromosome. Verification of construct expression was performed by immunohistochemistry. Follow-up electrophysiological studies revealed a significant reduction in γ-aminobutyric acid (GABA) release probability without alterations in postsynaptic membrane properties or changes in glutamatergic release probability in the prefrontal cortex pyramidal neurons. Behavioral characterization of our transgenic (Tg) mice uncovered that the Pvalb/Gad1 Tg mice have pronounced sensorimotor gating deficits, increased novelty-seeking and reduced fear extinction. Furthermore, NMDA (N-methyl-d-aspartate) receptor antagonism by ketamine had an opposing dose-dependent effect, suggesting that the differential dosage of ketamine might have divergent effects on behavioral processes. All behavioral studies were validated using a second cohort of animals. Our results suggest that reduction of GABAergic transmission from PVALB+ interneurons primarily impacts behavioral domains related to fear and novelty seeking and that these alterations might be related to the behavioral phenotype observed in schizophrenia.

  15. Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase

    DEFF Research Database (Denmark)

    Chen, Yun; Zhang, Yiming; Siewers, Verena


    Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported......Acetyl-coenzyme A (acetyl-CoA) is not only an essential intermediate in central carbon metabolism, but also an important precursor metabolite for native or engineered pathways that can produce many products of commercial interest such as pharmaceuticals, chemicals or biofuels. In the yeast...... into the cytoplasm or the mitochondria. However, whether acetyl-CoA generated in the mitochondria can be exported to the cytoplasm is still unclear. Here, we investigated whether the transfer of acetyl-CoA from the mitochondria to the cytoplasm can occur using a pyruvate decarboxylase negative, non...

  16. Structure of lpg0406, a carboxymuconolactone decarboxylase family protein possibly involved in antioxidative response from Legionella pneumophila. (United States)

    Chen, Xiaofang; Hu, Yanjin; Yang, Bo; Gong, Xiaojian; Zhang, Nannan; Niu, Liwen; Wu, Yun; Ge, Honghua


    Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all-helical fold with a conserved thioredoxin-like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate-binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.

  17. Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts. (United States)

    Nitta, Yoko; Yasukata, Fumiko; Kitamoto, Noritoshi; Ito, Mikiko; Sakaue, Motoyoshi; Kikuzaki, Hiroe; Ueno, Hiroshi


    Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

  18. The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases. (United States)

    Docimo, Teresa; Reichelt, Michael; Schneider, Bernd; Kai, Marco; Kunert, Grit; Gershenzon, Jonathan; D'Auria, John C


    Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of ¹³CO₂. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.

  19. Engineering salidroside biosynthetic pathway in hairy root cultures of Rhodiola crenulata based on metabolic characterization of tyrosine decarboxylase.

    Directory of Open Access Journals (Sweden)

    Xiaozhong Lan

    Full Text Available Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21-6.84, 1.50-2.19 and 1.27-3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.

  20. Variation in oxalate and oxalate decarboxylase production by six species of brown and white rot fungi

    DEFF Research Database (Denmark)

    Hastrup, Anne Christine Steenkjær; Oliver, Jason; Howell, Caitlin;

    cell lumen where it quickly dissociates into hydrogen ions and oxalate, resulting in a pH decrease of the environment, and oxalate-cation complexes. Generally, brown rot fungi accumulate larger quantities of oxalic acid in the wood than white rot fungi. The amount of oxalic acid has been shown to vary...... of formic acid and CO2 (Makela et al., 2002). So far only a few species of brown rot fungi have been shown to accumulate this enzyme (Micales, 1995, Howell and Jellison, 2006).   The purpose of this study was to investigate the variation in the levels of soluble oxalate and total oxalate, in correlation...

  1. Induction of YdeO, a regulator for acid resistance genes, by ultraviolet irradiation in Escherichia coli. (United States)

    Yamanaka, Yuki; Ishihama, Akira; Yamamoto, Kaneyoshi


    YdeO, an AraC-type transcription factor, is an important regulator in the induction of acid-resistance genes in Escherichia coli. In this study, we found that ydeO expression was induced 20 min after exposure to UV irradiation. This required the evgA and gadE genes in vivo. YdeO, induced by UV, controls the expression of a total of 21 genes. This accompanies SOS response in E. coli.

  2. Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene. (United States)

    Lasanajak, Yi; Minocha, Rakesh; Minocha, Subhash C; Goyal, Ravinder; Fatima, Tahira; Handa, Avtar K; Mattoo, Autar K


    S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene


    Directory of Open Access Journals (Sweden)

    E.S. Petrova


    Full Text Available Gamma-aminobutyric acid (GABA is a major inhibitory neurotransmitter in the central nervous system. Enzyme glutamate decarboxylase (GAD-67 is a marker of GABA-ergic neurons. The purpose of this study is to examine the distribution of GAD-67-immunopositive neurons in the striatum of rats under experimental conditions, reproducing brief focal cerebral ischemia. Endovascular occlusion of the left middle cerebral artery in rats was performed. Duration of circulatory disorders was 30 min, the time of reperfusion was 48 hours. With counting GAD-67-immunopositive neurons in the striatum was found that the number of GABA-ergic neurons in the striatum ipsilateral hemisphere is reduced by 40%. In the contralateral hemisphere, the distribution and structure of the neurons is not different from controls. It is shown that GABA-ergic neurons are less susceptible to damage, as compared to other neurons phenotypes.

  4. Multiplex PCR method for the simultaneous detection of histamine-, tyramine-, and putrescine-producing lactic acid bacteria in foods. (United States)

    Marcobal, Angela; de las Rivas, Blanca; Moreno-Arribas, M Victoria; Muñoz, Rosario


    In a screening of primers, we have selected three pairs of primers for a multiplex PCR assay for the simultaneous detection of lactic acid bacteria (LAB) strains, which potentially produce histamine, tyramine, and putrescine on fermented foods. These primers were based on sequences from histidine, tyrosine, and ornithine decarboxylases from LAB. Under the optimized conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases, a 924-bp fragment from tyrosine decarboxylases, and a 1,446-bp fragment from ornithine decarboxylases. When the DNAs of several target organisms were included in the same reaction, two or three corresponding amplicons of different sizes were observed. This assay was useful for the detection of amine-producing bacteria in control collection strains and in a LAB collection. No amplification was observed with DNA from nonproducing LAB strains. This article is the first describing a multiplex PCR approach for the simultaneous detection of potentially amine-producing LAB in foods. It can be easily incorporated into the routine screening for the accurate selection of starter LAB and in food control laboratories.

  5. Ghrelin and gastric acid secretion. (United States)

    Yakabi, Koji; Kawashima, Junichi; Kato, Shingo


    Ghrelin, a novel growth hormone-releasing peptide, was originally isolated from rat and human stomach. Ghrelin has been known to increase the secretion of growth hormone (GH), food intake, and body weight gain when administered peripherally or centrally. Ghrelin is also known to stimulate the gastric motility and the secretion of gastric acid. In the previous studies, the action of ghrelin on acid secretion was shown to be as strong as that of histamine and gastrin in in-vivo experiment. In the studies, the mechanism for the action of ghrelin was also investigated. It was shown that vagotomy completely inhibited the action of ghrelin on the secretion of gastric acid suggesting that vagal nerve is involved in the mechanism for the action of ghrelin on acid secretion. As famotidine did not inhibit ghrelin-induced acid secretion in the study by Masuda et al, they concluded that histamine was not involved in the action of ghrelin on acid secretion. However, we have shown that famotidine completely inhibited ghrelin-induced acid secretion and histidine decarboxylase (HDC) mRNA was increased in gastric mucosa by ghrelin injection which is inhibited by vagotomy Our results indicate that histamine is involved in the action of ghrelin on acid secretion. Furthermore synergistic action of gastrin and ghrelin on gastric acid secretion was shown. Although gastrin has important roles in postprandial secretion of gastric acid, ghrelin may be related to acid secretion during fasting period or at night. However, further studies are needed to elucidate the physiological role of ghrelin in acid secretion.

  6. Structure-function relations in oxaloacetate decarboxylase complex. Fluorescence and infrared approaches to monitor oxomalonate and Na(+ binding effect.

    Directory of Open Access Journals (Sweden)

    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

  7. Antiepileptic Potential of Matrine via Regulation the Levels of Gamma-Aminobutyric Acid and Glutamic Acid in the Brain

    Directory of Open Access Journals (Sweden)

    Jun Xiang


    Full Text Available Our present study aimed to determine the antiepileptic activity of matrine, and explore the possible molecular mechanism. To evaluate the antiepileptic activity of matrine, seizures in mice induced by PTZ and MES were established, then the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests in mice were also carried out. For the molecular mechanism investigations, contents of aspartic acid (Asp, gamma-aminobutyric acid (GABA, glutamic acid (Glu, glycine (Gly in seizures mice were determined; then, the chronic seizures rats induced by PTZ were prepared, and western blotting was used to determine the expressions of GAD 65, GABAA and GABAB in the brains. In the results, matrine showed significant antiepileptic effects on seizures mice induced by MES and PTZ. Moreover, the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests were also demonstrated that matrine had obvious antiepileptic effects. Additionally, our results revealed that after treatment with matrine, contents of GABA can be elevated, and the contents of Glu were obviously decreased. Furthermore, western blotting revealed that the mechanism regarding the antiepileptic effect of may be related to the up-regulations of GAD 65 and GABAA in the brain. Collectively, we suggested that matrine can be developed as an effective antiseptic drug.

  8. Antiepileptic potential of matrine via regulation the levels of gamma-aminobutyric acid and glutamic acid in the brain. (United States)

    Xiang, Jun; Jiang, Yugang


    Our present study aimed to determine the antiepileptic activity of matrine, and explore the possible molecular mechanism. To evaluate the antiepileptic activity of matrine, seizures in mice induced by PTZ and MES were established, then the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests in mice were also carried out. For the molecular mechanism investigations, contents of aspartic acid (Asp), gamma-aminobutyric acid (GABA), glutamic acid (Glu), glycine (Gly) in seizures mice were determined; then, the chronic seizures rats induced by PTZ were prepared, and western blotting was used to determine the expressions of GAD 65, GABAA and GABAB in the brains. In the results, matrine showed significant antiepileptic effects on seizures mice induced by MES and PTZ. Moreover, the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests were also demonstrated that matrine had obvious antiepileptic effects. Additionally, our results revealed that after treatment with matrine, contents of GABA can be elevated, and the contents of Glu were obviously decreased. Furthermore, western blotting revealed that the mechanism regarding the antiepileptic effect of may be related to the up-regulations of GAD 65 and GABAA in the brain. Collectively, we suggested that matrine can be developed as an effective antiseptic drug.

  9. Malonyl-CoA Decarboxylase (MCD) as a Potential Therapeutic Target for Breast Cancer (United States)


    of detection, using the ATP Bioluminescence Kit CLS II (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer’s protocol and read on a... ATP levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we s ynthesized a s mall m olecule i nhibitor o f M CD, 5...Figure 1). Acetyl-CoA carboxylase (ACC), the rate limiting enzyme of fatty acid synthesis, produces malonyl-CoA from the ATP dependent carboxylation of

  10. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Zheng Gong

    Full Text Available Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  11. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae. (United States)

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou


    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  12. Interneta sociālie tīkli, mammam/ un kā reklāmas nesēji Latvijā (2009.gads)



    Maģistra darba tēma ir „Interneta sociālie tīkli, mammam/ un kā reklāmas nesēji Latvijā (2009.gads)”. Darba mērķis ir izpētīt Latvijas interneta sociālo tīklu vidi kā potenciāli augošu un reklāmai piemērotu, kā arī izpētīt pamanāmākos reklāmas gadījumus šajos portālos. Teorijas daļā tiek apskatīta Web 2.0 ēra, interneta reklāmas un sociālo mediju mārketinga īpatnības, sociālo tīklu fenomens un teorijas, Latvijas reklāmas tirgus un auditorija, kā arī sociāl...

  13. Krīzes komunikācijas uzdevumi un metodes ārējā komunikācijā: Latvijas Krājbankas gadījuma analīze



    Bakalaura darba tēma ir „Krīzes komunikācijas uzdevumi un metodes ārējā komunikācijā: Latvijas Krājbankas gadījuma analīze”. Darba ietvaros tiek risināta pētījuma problēma – „Latvijas Krājbankā” realizētā ārējā komunikācija bankas krīzes gadījumā. Bakalaura darba ir strukturēts četrās nodaļās. Darba teorētiskajā bāzē tiek skaidroti krīzes, krīzes komunikācijas un ārējās komunikācijas jēdzieni. Darba empīriskajā daļā autore raksturojusi uzņēmumu „Latvijas Krājbanka” un pētījusi ārējās komun...

  14. Ghrelin and gastric acid secretion

    Institute of Scientific and Technical Information of China (English)

    Koji Yakabi; Junichi Kawashima; Shingo Kato


    Ghrelin, a novel growth hormone-releasing peptide, was originally isolated from rat and human stomach. Ghrelin has been known to increase the secretion of growth hormone (GH), food intake, and body weight gain when administered peripherally or centrally. Ghrelin is also known to stimulate the gastric motility and the secretion of gastric acid. In the previous studies, the action of ghrelin on acid secretion was shown to be as strong as that of histamine and gastrin in-vivo experiment. In the studies, the mechanism for the action of ghrelin was also investigated. It was shown that vagotomy completely inhibited the action of ghrelin on the secretion of gastric acid suggesting that vagal nerve is involved in the mechanism for the action of ghrelin on acid secretion. As famotidine did not inhibit ghrelin-in-duced acid secretion in the study by Masuda et al, they concluded that histamine was not involved in the action of ghrelin on acid secretion. However, we have shown that famotidine completely inhibited ghrelin-induced acid secretion and histidine decarboxylase (HDC) mRNA was increased in gastric mucosa by ghrelin injection which is inhibited by vagotomy Our results indicate that histamine is involved in the action of ghrelin on acid secretion. Furthermore synergistic action of gastrin and ghrelin on gastric add secretion was shown. Although gastrin has important roles in postprandial secretion of gastric acid, ghrelin may be related to acid secretion during fasting period or at night. However, further studies are needed to elucidate the physiological role of ghrelin in acid secretion.

  15. GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

    Energy Technology Data Exchange (ETDEWEB)

    Kowalski, Antoni, E-mail: [Department of Molecular Neurochemistry, Medical University of Lodz, 6/8 Mazowiecka Str., 92-215 Lodz (Poland); Zylinska, Ludmila, E-mail: [Department of Molecular Neurochemistry, Medical University of Lodz, 6/8 Mazowiecka Str., 92-215 Lodz (Poland); Boczek, Tomasz, E-mail: [Department of Molecular Neurochemistry, Medical University of Lodz, 6/8 Mazowiecka Str., 92-215 Lodz (Poland); Rebas, Elzbieta, E-mail: [Department of Molecular Neurochemistry, Medical University of Lodz, 6/8 Mazowiecka Str., 92-215 Lodz (Poland)


    Highlights: {yields} Suppression of PMCA2 or PMCA3 slows down proliferation of GH3 cells. {yields} PMCA2 suppression lowers the activity of GABA-shunt enzymes. {yields} PMCA3 suppression increases the expression of glutamate decarboxylase 65. {yields} PMCA2 and PMCA3 function appears to be linked to regulation of GABA metabolism. -- Abstract: GABA ({gamma}-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.

  16. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations. (United States)

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


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

  17. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea. (United States)

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen


    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  18. 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.

  19. QSAR study of malonyl-CoA decarboxylase inhibitors using GA-MLR and a new strategy of consensus modeling. (United States)

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


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

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

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


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