Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Relevance of adenosine deaminase as a marker for tuberculous pleural effusion in developing countries

Objective:Relevance of estimation of pleural adenosine deaminase(PADA) and serum adenosine deaminase (SADA) levels in-pleural effusion especially in cases of lymphocytic predominant exudative tubercular effusions. Methods:Fifty patients(33 male and 17 female;age:44.12±11.51 years) with pleural effusions were selected to assay adenosine deaminase(ADA) activity in pleural fluid and serum in adjunct to pleural fluid analysis.Effusions were individually classified as transudates or exudates after careful evaluation of all the biochemical parameters of pleural fluid and serum of patients and on the basis of Lights criteria.Cutoff value for PADA was taken as 60U/L and that for pleural/serum ADA ratio(P/S ADA) was 1.8.Results:Fourty -three patients had exudative effusions among which 38 patients had tuberculous pleural effusions and 5 had nontubercular effusions.7 cases were transudates.Mean PADA levels in tubercular group(78.95±25.32 U/ L) were found to be much higher P=0.0000) than nontubercular(23.00±5.22 U/L) group.SADA levels in tubercular group(31.05±6.42 U/L) were significantly higher(P=0.0000)as compared to nontubercular group(15.58±8.35 U/L).PADA cutoff at 60 U/L yielded sensitivity and.specificity of 81.5%and 100%respectively,whereas P/S ADA ratio at 1.8 gave sensitivity and specificity of 84.2% and 75%respectively. A positive correlation(r=0.507,P= 0.001 1)between PADA and SADA was found in tubercular group but no such correlation(r=0.302,P=0.3407)was observed in nontubercular group.Conclusion: The measurement of ADA in tubercular pleural effusions has not only relevance but also a high diagnostic utility when other clinical and laboratory tests are either negative or confusing.

Relationship between serum adenosine deaminase levels and liver histology in autoimmune hepatitis

AIM To evaluate the relationship between serum adenosine deaminase(ADA) levels and histological features in patients with autoimmune hepatitis(AIH).METHODS A total of 80 subjects(52 AIH cases and 28 healthy controls) were included in the study. Patients were diagnosed according to the simplified criteria suggested by the International Autoimmune Hepatitis Group. All of the cases had been diagnosed with AIH between 2010-2015 at Hacettepe University, Department of Gastroenterology. Serum blood samples were collected and stored at-80 ℃ until the biochemical estimation of ADA activity. The diagnosis of patients was confirmed by liver biopsy. Serum ADA > 20 U/L was considered to be high level.RESULTS Mean serum ADA levels were significantly higher in AIH patients than those in healthy controls(25.4 ± 9.6 U/L vs 12.8 ± 2.2 U/L, P < 0.001). Serum ADA levels > 20 U/L were found in 63.5% AIH patients and in 0% healthy controls(P < 0.001). Mean serum ADA levels were significantly increased in each stage of histological activity: 15.2 ± 3.5 U/L for patients with mild interface hepatitis, 23.1 ± 10.0 U/L for patients with moderate interface hepatitis and 30.9 ± 7.0 U/L for patients with severe interface hepatitis(P < 0.001). Correlation analysis showed that there was a positive association between serum ADA levels and histological activity(r = 0.71, P < 0.001). Receiver operating characteristic analysis suggested that 24.5 U/L was the optimum cut-off point of ADA level for severe interface hepatitis(sensitivity 88%, specificity 85.2%, area under thecurve: 0.88).CONCLUSION Because of the positive correlation with inflammatory activity, serum ADA level may be a potential biomarker for predicting or monitoring histological activity in patients with AIH.

Role of ascites adenosine deaminase in differentiating between tuberculous peritonitis and peritoneal carcinomatosis

AIM： To investigate the usefulness of tumor markers and adenosine deaminase in differentiating between tuberculous peritonitis （TBP） and peritoneal carcinoma- tosis （PC）. METHODS： A retrospective analysis of data was per- formed on consecutive patients who underwent perito- neoscopic and abdominal computed tomography （CT） evaluations. Among 75 patients at the Seoul National University Hospital from January 2000 to June 2010 who underwent both tests, 27 patients （36.0%） and 25 patients （33.3%） were diagnosed with TBP and PC, re- spectively. Diagnosis was confirmed by peritoneoscopic biopsy. RESULTS： Serum c-reactive protein （7.88：1：6.62 mg/ dL vs 3.12 ＋ 2.69 mg/dL, P = 0.01）, ascites adenos- ine deaminase （66.76：1：32.09 IU/L vs 13.89 ：l： 8.95 IU/L, P 〈 0.01）, ascites lymphocyte proportion （67.77 ：1： 23.41% vs 48.36 ＋ 18.78%, P 〈 0.01）, and serum- ascites albumin gradient （0.72 ＋ 0.49 g/dL vs 1.05 ＋ 0.50 g/dL, P = 0.03） were significantly different be- tween the two groups. Among tumor markers, serum and ascites carcinoembryonic antigen, serum carbohy- drate antigen 19-9 showed significant difference be- tween two groups. Abdominal CT examinations showed that smooth involvement of the parietal peritoneum was more common in the TBP group （77.8% vs 40.7%） whereas nodular involvement was more common in the PC group （14.8% vs 40.7%, P = 0.04）. From receiver operating characteristic （ROC） curves ascites adeno- sines deaminase （ADA） showed better discriminative capability than tumor markers. An ADA cut-off level of 21 IU/L was found to yield the best results of differ- ential diagnosis; sensitivity, specificity, positive predic- tive value, and negative predictive value were 92.0%, 85.0%, 88.5% and 89.5%, respectively. CONCLUSION： Besides clinical and radiologic findings, ascitic fluid ADA measurement is helpful in the differen- tial diagnosis of TBP and PC.

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-l were recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis

L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

Adenosine deaminase isoenzymes estimation - as a diagnostic tool for tuberculous pleural effusions

Objective:To assess the efficacy of ADA isoenzyme estimation over that of total ADA level in pleural fluid and serum as a more efficient diagnostic indicator in tuberculous pleural effusions in high prevalence country like India.Methods:The efficacy was analysed in total thirty four patients of pleural effusions.Total ADA was estimated by Guitsi and Galanti Calorimetric method and ADA isoenzymes with and without EHNA[Erythro-9-（2- hydroxy-3-nonyl） adenine]a potent ADA1 inhibitor using the same method.Results:The results demonstrated a statistically significant values of ADA2 in serum（P【0.001）,pleural fluid（P = 0.000） and significant value for the ratio of pleural fluid ADA2/serum ADA2（P【0.001） and pleural fluid ADA/ADA(2（P【0. 005）.The sensitivity and specificity values of pleural fluid ADA|2 is 81.8%and 91.6%（cut off value 60 IU/L for Tuberculous effusions）,serum ADA2 95.4%and 66%（cut off value 70 IU/L for tuberculous effusions）. ADA2 is an isoenzyme,which is significantly raised in tuberculous pleural effusions both in the serum and pleural fluid.Conclusion:Estimation of ADA isoenzymes is redundant as a diagnostic aid over total ADA estimation in view of the limited improvements both in specificity and sensitivity patterns and also in term of cost-benefit ratio.

Expression of RNA Editing Deaminase on Human Glioma Cell Lines

To study the expression of RNA editing deaminases ADAR2 and ADAR3 in different malignant glioma cell lines and the effect of phenylacetate on the expression of these genes, the primarily glial cells of human brain tissue were isolated and cultured. The human glioma SHG-44, U-251, BT-325 cell lines were maintained in culture. The expressions of ADAP,.2 and ADAR3 mRNA were detected by the semiquantitative reverse transcription-polymerase chain reaction（RT-PCR）. The changes in ADAR2 mRNA expression before and after phenylacetate treatment were detected by RT-PCR and image analysis. The level of ADAR gene expression is expressed as the ratio expression rate（RER） of ADAR gene to β-actin according to computer image analysis. ADAR2 displays moderate expression in glial cells, low expression in low-grade malignant glioma SHG-44 cells, and high level expression in high-grade malignant glioma U-251and BT-325 cells. The expression of ADAR2 can be decreased by phenylacetate treatment in glioma U-251 cells. ADAR3 is not expressed in normal brain glial cells, or glioma SHG-44, U-251 and BT-325 cells before and after phenylacetate treatment. The enhanced expression of ADAR2 may be involved in the tumor progression of malignant glioma. Phenylacetate can decrease the expression of ADAR2 in glioma cells, suggesting that it may act on the RNA editing process in glioma.

Adenovirus-mediated tissue specific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression level

Hepatocellular carcinoma (HCC) is one of the mostcommon cancers in the world, especially in East Asia.There is no standardized or effective strategy could beadapted routinely except of some early diagnosedpatients, and the prognosis is poor. In recent years, genetherapy has become a standard experimental approach fortreating cancers that have escaped conventionaltherapies. One such an approach is to confer the tumorcells with sensitivity to chemical reagents through

Inhibitory effects of cytosine deaminase gene-transfected bone marrow mesenchymal stem cells on glioma cell proliferation

Bone marrow mesenchymal stem cells were isolated from C57BL mice, transfected with the cytosine deaminase （CD） gene using a lentivirus vector and co-cultured with C6 glioma cells to verify anti-tumor effects of bone marrow mesenchymal stem cells carrying CD genes. C57MSC-CD/eGFP cells converted 5-fluorocytosine to 5-fluorouracil and exhibited significant inhibition of proliferation and apoptosis in C6 glioma cells. C57MSC-CD/eGFP cells were then implanted into rat models of brain C6 glioma. Rats were also intraperitoneally injected with 5-fluorocytosine after 7 days. MSC-CD/eGFP cells were irregularly distributed at the margin of the glioma, as well as encased and reduced the volume of the glioma. CD-transfected bone marrow mesenchymal stem cells inhibit the in vivo growth and in vitro proliferation of glioma.

Migration to gliomas of bone mesenchymal stem cells transfected with cytosine deaminase gene following transplantation in vivo

This experiment sought to observe the migration and distribution of bone mesenchymal stem cells transfected with the cytosine deaminase gone （BMSCs-CD/eGFP） after transplantation in vivo through three pathways. In addition, we examined the tropism of these cells to glioma. Intracranial C6 glioma models were established in Sprague-Dawley rats using an intracranial stereotactic inoculation method. When tumors were 7 days old, rats were inoculated with lx106 BMSCs-CD/eGFP cells via the tumor-bearing internal carotid artery, the contralateral hemisphere and the tumor-bearing glioma. Fluorescence microscopy revealed that BMSCs-CD/eGFP exhibited a strong capacity for migration to tumors. BMSCs-CD/eGFP transplanted via the tumor-bearing intemal carotid artery were observed to distribute in glioma tissues. BMSCs-CD/eGFP inoculated via the ipsilateral glioma mainly located within and at the edge of glioma tissues. BMSCs-CD/eGFP inoculated via the contralateral hemisphere mainly distributed at the proximal end of the tumor at the incubation site.

Combined Role of ACC Deaminase Producing Bacteria and Biochar on Cereals Productivity under Drought

Most of the cereal crops are widely cultivated to fulfil the humans food requirements.Under changing climate scenario,the intensity of drought stress is continuously increasing that is adversely affecting the growth and yield of cereal crops.Although the cereals can tolerate moderate drought to some extent,but mostly they are susceptible to severe drought stress.Higher biosynthesis of ethylene under drought stress has been reported.Many scientists observed that inoculation of 1-aminocyclopropane-1-carboxylate(ACC)deaminase producing plant growth promoting rhizobacteria(PGPR)is an efficacious tool to overcome this problem.These PGPR secrete ACC deaminase which cleavage the ACC into the compounds,other than ethylene.Furthermore,secretion of growth hormones also play imperative role in enhancing the growth of the cereals under limited availability of water.In addition,the use of biochar has also been recognized as another effective amendment to grant resistance against drought.Biochar application improves the soil physiochemical attributes i.e.,porosity,nutrients retention and water holding capacity which decrease the loss of water and increase its bioavailability.In recent era,the idea of coapplication of ACC deaminase producing PGPR and biochar is becoming popular which might be more efficient to use water under drought stress.The aim of current review is to combine the facts and understanding of this novel idea to grant maximum resistance to crops against drought stress.Some scientists have observed significant improvement in yield of cereal crops by combined use of ACC deaminase producing PGPR and biochar.However,more research is suggested for deep understanding of complex synergistic mechanism of ACC deaminase activity in combination with biochar.

Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu8988

Objective:To evaluatethein vitro killingeffectsof cytosinedeaminasegene mediatedby adenovirusvector on humanpancreaticcarcinoma.Methods:CytosineDeaminase(CD)genewas clonedintopAdTrack-CMV-CD,pAd-Track-CMV-CDandpAdEasy-1wererecombinedinbacteria,andtheproductscontaininggreenfluorescentprotein(GFP)werepropagatedin293cellsandpurifiedby cesiumchloridegradientcentrifugation.Humanpancreaticcarcinomacellline8988wereinfectedwiththisvirus,then5-FCwasadded;XTTassaywasusedto estimatetherelativenumbersof viable cells.Results:Thepositivecloneswereconfirmedby usingendonucleasedigestion,andthetiterof theviruscontaining CD genewas2×10 11 pfu/ml.Itwasfoundthat5-FCpossessedsignificantcytotoxicactivitiesforCD genetransfected8988cellline,buthadlittleeffectson non-transfectedpancreaticcarcinomacells.Conclusion:CD genemediatedby adenovirus hasa highinfectivityandis efficientforkillingculturedpancreaticcarcinomacells,indicatingsuicidegenemaybe effec-tiveforpancreaticcancerinfuture.

Protection of a cytidine deaminase gene gainst toxicity of high dose chemotherapy in mice

Objective： To explore the feasibility of transfecting cytidine deaminase （CD） gene into mouse bone marrow cells in order to observe the drug resistance of high dose Ara-C and improve the tolerance of myelosuppression following combination chemotherapy. Methods： Human cytidine deaminase gene was transfected into mice bone marrow cells by retroviral vector. Resistant colony-forming unit granulocyte-macrophage （CFU-GM） assay was performed after the transfected mice bone marrow cells treated by the Ara-C. DNA was extracted from mice bone marrow cells. The drug resistant gene in mice bone marrow cells after transfection was detected by PCR. Results： Bone marrow cells of the donor mice cultured with the retroviral producer cells showed the drug resistant colonies and resistance to Ara-C, so did accept mice transplanted with the CD gene （CFU-GM of donor mice was 52%, χ^2 = 124.62, P 〈 0.01; accept mice was 54%, χ^2 = 126.26, P 〈 0.01, both compared with the contrast group）. The animal survival rate was significantly higher in gene transfected group than that of the control （χ^2= 7.42, P 〈 0.01）. CD gene of transfected bone marrow cells was confirmed by PCR. Conclusion： CD gene can be transfected into bone marrow cells of mice efficiently and increase the drug resistance to Ara-C.

Purification and characterization of <i>Aspergillus parasiticus</i>cytosine deaminase for possible deployment in suicide gene therapy

Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax gave pKa1 of 5.8 and pKa2 of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca2+, Co2+, Zn2+ and Hg2+ and completely inhibited by Cu2+ and Fe2+ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.

Interaction of Cationic and Anionic Phthalocyanines with Adenosine Deaminase, Molecular Dynamics Simulation and Docking Studies

Interactions of anionic, cationic and metal phthalocyanine with adenosine deaminase were studied by molecular dynamics and docking simulation. Structural parameters such as solvent accessible surface area (SAS), mid-point of transition temperature (Tm), radial distribution function (RDF) and hydrogen bond, helix, coil, beta percentage and other physical parameters were obtained. The denaturation of adenosine deaminase (ADA) by heat, anionic and cationic phthalocyanines was compared. A series of 20 ns simulation performed at temperatures ranging from 275 to 450 K, starting from the ADA native structure. Results of radial distribution functions (RDFs) showed that metallic derivative at low concentration behaves the same as osmolytes that increases the beta form and increases the enzyme stability. Molecular docking studies have been carried out to confirm the simulation results. Investigation of binding site and free energy confirmed that the efficiency of interaction with adenosine deaminase depends on metal core. Binding energy of non-metallic form is more negative than metallic form and it significantly decreases for phthalocyanine. Self-aggregation of anionic phthalocyanine decreases in comparison with cationic derivative, therefore enzyme denaturation in the presence of anionic form is higher than the other. Furthermore, thermal stability of the enzyme also depends on temperature in presence of phthalocyanine. Binding site of phthalocyanine on the enzyme has been identified by docking analysis.

Plant Growth Promoting Rhizobacteria Having 1-Aminocyclopropane-1-Carboxylic Acid Deaminase to Induce Salt Tolerance in Sunflower (<i>Helianthus annus L.</i>)

Soil salinity badly affects agriculture productivity through accumulation of salts in upper layers of soils. The harmful effects of salts in arable lands have influenced modern as well as ancient civilizations. A pot study was carried out to test the performance of two PGPR isolates (KS 8, KS 28) on sunflower (SMH-0917) under different salinity levels (8, 10 and 12 dS·m-1). These salinity levels were developed by adding calculated amount of salts (NaCl, Na2SO4, CaCl2 and MgSO4) with ratio of 3:4:2:1. The bacterial strains KS 8 and KS 28 were applied separately in two treatments while third treatment was co-inoculation (KS mix). Completely randomized experimental design (CRD) was used and data were collected at flowering stage about pre-decided plant growth parameters (plant height, shoot dry weight and root dry weight). The bacterial isolate KS 8 showed an increase of 26, 102% and 83% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while this improvement was 67%, 163% and 296% at EC 10 dS·m-1, however an increase of 100%, 74% and 382% was recorded over control respectively at EC 12 dS·m-1. Similarly isolate KS 28 exhibited an increase of 14%, 69% and 54% in plant height;shoot dry weight and root dry weight at EC 8 dS·m-1, whereas this improvement was 56%, 163% and 188% at EC 10 dS·m-1, while an increase of 60%, 41% and 282% was registered respectively over control at EC 12 dS·m-1. The increase due to mixture treatments was 4%, 41% and 16% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while an increase of 33%, 57% and 100% at EC 10 dS·m-1, whereas an improvement of 53%, 33% and 164% respectively was noted at EC 12 dS·m-1 over un-inoculated. The isolate KS 8 performed better than KS 28 and mixture treatment. These two PGPR strains could be used to mitigate the adverse impact caused by salinity stress on sunflower.

Changes in Adenosine Metabolism in Asthma. A Study on Adenosine, 5&#39-NT, Adenosine Deaminase and Its Isoenzyme Levels in Serum, Lymphocytes and Erythrocytes

Background: Adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) play a crucial role in adenosine metabolism in healthy individuals. Adenosine is an inflammatory mediator of asthma. Changes in adenosine metabolism and role of ADA and 5'-NT in regulating adenosine level in asthmatics and correlation of these changes with severity of asthma are not clearly understood. Methods: In this study, we screened 5217 patients, of which 2416 were diagnosed with asthma. Further, of 2416 asthmatics, only 45 patients who strictly fulfilled the selection criteria were enrolled in the study. The patients were classified into mild, moderate and severe persistent groups;each group consisted of fifteen patients. Fifteen healthy subjects served as controls. Adenosine levels and activities of 5'-NT, total ADA, ADA1 and ADA2 in serum, lymphocytes and erythrocytes were determined. The data were analysed statistically and p vice versa.

Biosynthesis of 4-hydroxyphenylpyruvic acid from L-tyrosine using recombinant Escherichia coli cells expressing membrane bound L-amino acid deaminase

4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to prod uce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase （mL-AAD）, which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide （twin-arginine translocation pathway, Tat） and integrated pelB （the general secretory pathway, Sec）-Tat signal peptide was determined and compared firstly, using two pET systems （pET28a and pET20b）. It was found that the Tat pathway （pET28a-mlaad） resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts BI21 （DE3） and 0.05 mmol. L- 1 IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol,L 1 4-HPPA was obtained from 100 mmol.L 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.