Expression of Recombinant Tryptophan Decarboxylase in Different Subcellular Compartments in Tobacco Plant

The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.

Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

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 purification 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℃, respectively. The purified GAD had a half-life of 50rain at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22.5＇-pyridoxal phosphate （PLP） had little effect on the regulation of its activity.

Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

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.

Expression of ornithine decarboxylase in precancerous and cancerous gastric lesions

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 well-differentiated 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.

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

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 °C. 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.

Bile acid increases expression of the histamine-producing enzyme,histidine decarboxylase,in gastric cells

AIM: To investigate the effect of bile acid on the expression of histidine decarboxylase (HDC), which is a major enzyme involved in histamine production, and gene expression of gastric transcription factors upon cooperative activation.

Effect of remedies of supporting resistance and relieving blood stasis on metastasis of postoperative gastric cancer and ornithine decarboxylase

AIM To study the action of remedies of supporting resistance and relieving blood stasis on metastasis of postoperative gastric cancer and its influence on ornithine decarboxylase (ODC). METHODS Sixty three postoperative gastric cancer patients were randomly divided into two groups. Thirty one patients were treated with western medicine consisting of the FAP scheme (5 flurouracil, adriamycin and cisplatin) and the CODP scheme (cyclophosphamide, vincristine, daunorubicin and prednisone), whereas 32 patients with the FAP scheme and traditional Chinese medicine. Correlations were approached between the ODC levels detected before and after treatment and some other factors including tumor diameter, infiltration depth, histological type and lymph node metastasis. RESULTS The ODC levels in the gastric cancers and normal gastric mucosa adjacent to the cancer tissues were significantly higher in the patients than those of the controls. The increasing ODC was obviously correlated with the tumor size, infiltration depth, differentiation type, and lymph node metastasis. Six months later there was no significant changes in the ODC levels of the group using only western medicine, while the ODC levels decreased markedly in the group using combined western and traditional Chinese medicine ( P <0 01). CONCLUSION The remedies of traditional Chinese medicine on the metastases of postoperative gastric cancers were related to the reduction of ODC activities.

REVERSION OF MALIGNANT PHENOTYPES OF HUMAN LUNG SQUAMOUS CARCINOMA CELLS BY ORNITHINE DECARBOXYLASE ANTISENSE RNA

Abnormally elevated activity of ornithine decarboxylase (ODC), and subsequent polyamine accumulation are intimately associated with the genesis.development and metastasis of cancer. In the present study, to control the growth of tumor cells, ODC antisense RNA was used to transfect human lung squamous carcinoma cell line LTEP-78. Compared with the parental cells, growth of the antisense transfected LTEP-78 cells arrested in G0/Gl phase and colony formation in soft agarose and tumorigenicity in nude mice were significantly reduced. Nucleic acid hybridization demonstrated that the transfectants expressed a high level of ODC antisense RNA and a significantly reduced level of endogenous ODC mRNA.The results suggest that the reversion of malignant phenotypes of human lung squamous carcinoma cells transfected with ODC antisense RNA is associated with the inhibition of polyamine biosynthesis.

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.

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

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.

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

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.

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

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.

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

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, 21 and 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.

Stiff-Person Syndrome Associated with Anti-Glutamic Acid Decarboxylase Autoimmune Encephalitis in a Young Woman: A Case Report

A 34-year-old female with stiff-person syndrome(SPS)is reported in this paper.She experienced short-term memory impairment and was diagnosed with anti-glutamic add decarboxylase(GAD)autoimmune encephalitis(AE)at the local hospital.However,after the treatment with intravenous immunoglobulin and highdose glucocorticoids,her symptoms unchanged.Two months later,she was admitted to our hospital due to an unstable gait and persistent leg stiffness,at which point she was diagnosed as anti-GAD AE concomitant with SPS.Her clinical symptoms improved with an increased dose of y-aminobutyric acid(GABA)-enhancing drug and plasma exchange.Anti-GAD antibody-associated AE combined with SPS is extremely rare.Treatment with GABA-enhancing drugs and appropriate immunotherapy can improve the neurological function of patients suffering from the combination of SPS and limbic encephalitis.

Characterization and application of a recombinant dopa decarboxylase from Harmonia axyridis for the efficient biosynthesis of dopamine

Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.

Aromatic L-amino acid decarboxylase cells in the spinal cord:a potential origin of monoamines

Monoamine neurotransmitters include catecholamines and indoleamines. The most common catecholamines are do- pamine （DA）, noradrenaline （NA） and adrenaline, which are produced from phenylalanine and tyrosine; whereas the most common indoleamine is serotonin （5-hydroxytrypta mine, 5-HT）, which is produced from 5-hydroxytryptophan （5-HTP）.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours

AIM: To study histidine decarboxylase(HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus(n = 3) and corpus(n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours(GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2(VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid(U-Me Im AA) was determined using highperformance liquid chromatography in 27 of the 64 patients.RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cellpopulation co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-Me Im AA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Coexpression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

The Inhibitory Effect of Curcumin on Ornithine Decarboxylase against Hepatic Carcinoma

Curcumin the active component of turmeric is widely used as an anticancer agent for treating many human cancers. This study aimed at the extraction of curcumin from Curcuma Longa and investigates its therapeutic effect as ornithine decarboxylase (ODC) inhibitor in HepG2 cells. The proliferation of HepG2 cells was carried out by using the MTT assay. In addition, cell cycle analysis was evaluated by using the flow-cytometric technique. Our results showed that curcumin has the ability to inhibit the proliferation of HepG2 cells with IC50 of 24.79 μg/ml and induced G2/M cell cycle arrest. Moreover, it caused an elevation in the intracellular concentration of Ca2+. Moreover, in the curcumin administration the downregulation expression level of ODC and Bcl-2 genes (p ≤ 0.05) was significant found. On the other hand, upregulation in the expression level of P53, Bax, and caspase-3 genes (p ≤ 0.05). This study concluded that curcumin may be considered as a new saving candidate for the future progress of antitumor agents.

A potential energy profile of the catalytic cycle of pyruvate decarboxylase

A computational study on the mechanism for the decarboxylation of pyruvic acid to acetaldehyde catalyzed by pyruvate decarboxylase at the B3LYP/6-31G (d, p) level of theory is presented. The model employed is self-contained and it does not resort to external groups to provide protons to the various structures in the mechanism. The potential energy surface points at the intramolecular proton transfer from the amino group of the pyrimidine ring in the enamine intermediate to the enol exocyclic carbon as the rate-determining step (with a barrier of 20.55 kcal·mol–1). This value is in reasonable agreement with an estimated barrier of 24.76 kcal·mol–1, derived from the experimental rate constant (4.0 10–5 s–1) for the decarboxylation of α-lactylthiamin.

RAPID DETERMINATION OF L-GLUTAMIC ACID WITH AN ENZYME REACTOR OF L-GLUTAMIC DECARBOXYLASE IMMOBILIZED ON ION EXCHANGE RESIN

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 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 1.0×10-5 M. The linearity response is in the range of 5×10 -2-5×10 -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%.