A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

KAUST Repository

La, Honggui

2011-09-06

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

Energy Technology Data Exchange (ETDEWEB)

Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

2014-05-15

Highlights: • Inhibition of H{sub 2}S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H{sub 2}S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H{sub 2}S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H{sub 2}S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H{sub 2}S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H{sub 2}S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

Crystallization and preliminary X-ray analysis of βC–S lyases from two oral streptococci

International Nuclear Information System (INIS)

Kezuka, Yuichiro; Yoshida, Yasuo; Nonaka, Takamasa

2009-01-01

The βC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, were cloned, overproduced, purified and crystallized. The obtained crystals were characterized by X-ray diffraction. Hydrogen sulfide, which causes oral malodour, is generally produced from l-cysteine by the action of βC–S lyase from oral bacteria. The βC–S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus βC–S lyase belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 Å, and the crystal of S. gordonii βC–S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 Å. The structures of the βC–S lyases were solved by molecular-replacement techniques

Cloning and knockout of formate hydrogen lyase and H{sub 2}-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Zhao, Hongxin; Ma, Kun; Lu, Yuan; Zhang, Chong; Wang, Liyan; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084 (China)

2009-01-15

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A ({delta} hycA), IAM1183-O ({delta} hybO), and IAM1183-AO ({delta} hycA/ {delta} hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 {+-} 38.59, 2747.203 {+-} 13.25 and 3372.019 {+-} 4.39 (ml h{sup -1} g{sup -1}dry cell weight), respectively, higher than that of the wild strain (2321.861 {+-} 15.34 ml h{sup -1} g{sup -1}dry cell weight). The total H{sub 2} yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H{sub 2}/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H{sub 2}/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183 {delta} hycA/ {delta} hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L. (author)

Correlation between the cystathionine-r-lyase (CES) and the ...

African Journals Online (AJOL)

Background: The infection of Helicobacter pylori (H. pylori) is one of the most important causes of gastric ulcer disease. The role of hydrogen sulfide (H2S) production in H. pylori-induced gastric ulcer disease. Aim: The expression of cystathionine-γ-lyase (CSE) was determined, and correlated with the severity of gastric ulcer ...

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

Science.gov (United States)

Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

2015-11-01

Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning and study of the pectate lyase gene of Erwinia carotovora

International Nuclear Information System (INIS)

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.; Syarinskii, M.A.; Strel'chenko, P.P.; Yankovski, N.K.; Alikhanyan, S.I.; Fomichev, Yu.K.; Debabov, V.G.

1986-01-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector λ 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representative gene libraries on phage vectors from no less than 1 μg of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, λ 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

Science.gov (United States)

Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

2011-08-01

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

International Nuclear Information System (INIS)

Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria

2006-01-01

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl 2 , as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

International Nuclear Information System (INIS)

Mishra, Jayshree; Khurana, Seema; Kumar, Narendra; Ghosh, Ananta K.; Das, Debabrata

2004-01-01

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H 2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster

Crystallization and preliminary X-ray analysis of argininosuccinate lyase from Streptococcus mutans

International Nuclear Information System (INIS)

Cao, Yan-Li; Li, Gui-Lan; Wang, Kai-Tuo; Zhang, Hong-Yin; Li, Lan-Fen

2011-01-01

Crystals of argininosuccinate lyase from S. mutans were obtained and X-ray data were collected to 2.5 Å resolution in space group R3. Argininosuccinate lyase (ASL) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. The gene encoding Streptococcus mutans ASL (smASL) was amplified and cloned into expression vector pET28a. The recombinant smASL protein was expressed in a soluble form in Escherichia coli strain BL21 (DE3) and purified to homogeneity by two-step column chromatography. Crystals suitable for X-ray analysis were obtained and X-ray diffraction data were collected to a resolution of 2.5 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 254.5, c = 78.3 Å

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

DEFF Research Database (Denmark)

Jochimsen, Bjarne; Lolle, Signe; McSorley, Fern R.

2011-01-01

Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed...

Structural insights into the bacterial carbon - phosphorus lyase machinery

DEFF Research Database (Denmark)

Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten

2015-01-01

Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon......–phosphorus (C–P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C–P lyase core complex (PhnG–PhnH–PhnI–PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero...

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

International Nuclear Information System (INIS)

Xu, H. Howard; Real, Lilian; Bailey, Melissa Wu

2006-01-01

With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats

Revolutionizing membrane protein overexpression in bacteria

NARCIS (Netherlands)

Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory,

Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

Directory of Open Access Journals (Sweden)

Khan MA

2013-04-01

Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses

DEFF Research Database (Denmark)

Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

2016-01-01

Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short...

GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

Science.gov (United States)

Goyal, Megha; Chaudhuri, Tapan K

2015-07-01

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger

DEFF Research Database (Denmark)

Meijer, Susan Lisette; Otero, José Manuel; Olivares Hernandez, Roberto

2009-01-01

of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate...... towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct...

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

Science.gov (United States)

Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

NARCIS (Netherlands)

Boxma, B.; Voncken, F.L.M.; Jannink, S.A.; Alen, T.A. van; Akhmanova, A.S.; Weelden, S.W. van; Hellemond, J.J. van; Ricard, G.N.S.; Huynen, M.A.; Tielens, A.G.; Hackstein, J.H.P.

2004-01-01

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of

Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

Science.gov (United States)

Zheng, Ren-Chao; Tang, Xiao-Ling; Suo, Hui; Feng, Li-Lin; Liu, Xiao; Yang, Jian; Zheng, Yu-Guo

2018-05-01

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg -1 . Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, β-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the k cat /K m value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL -1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA. Copyright © 2017 Elsevier Inc. All rights reserved.

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

International Nuclear Information System (INIS)

Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena; Indiveri, Cesare

2012-01-01

Highlights: ► mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. ► The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. ► The protein solubilised in Triton X-100 has been reconstituted in liposomes. ► Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni 2+ -chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [ 3 H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K m for carnitine was 36 μM. [ 3 H]carnitine transport was inhibited by carnitine analogues and some Cys and NH 2 reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

Dark fermentative hydrogen and ethanol production from biodiesel waste glycerol using a co-culture of Escherichia coli and Enterobacter sp.

NARCIS (Netherlands)

Maru, B.T.; López, F.; Kengen, S.W.M.; Constantí, M.; Medina, F.

2016-01-01

In previous comparative studies, Enterobacter spH1 was selected as the best hydrogen and ethanol producer (Knothe, 2010). Here, glycerol fermentation was compared between three other strains: Escherichia coli CECT432, Escherichia coli CECT434 and Enterobacter cloacae MCM2/1. E. coli CECT432 was

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase.

Science.gov (United States)

Sugimura; Sawabe; Ezura

2000-01-01

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596(T), which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2.

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

Science.gov (United States)

Roberts, D P; Berman, P M; Allen, C; Stromberg, V K; Lacy, G H; Mount, M S

1986-07-01

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.

Impact of different alginate lyases on combined cellulase–lyase saccharification of brown seaweed

DEFF Research Database (Denmark)

Manns, Dirk Martin; Nyffenegger, Christian; Saake, B.

2016-01-01

-guluronic acid. When applied together with a fungal cellulase preparation (Cellic®CTec2) at pH 6 and 40 °C on a glucan rich brown seaweed Laminaria digitata the viscosity decreased in the initial minutes while measurable alginate degradation occurred primarily within the first 1–2 hours of reaction. Whereas FALy......, indicating that the degradation of mannuronic acid blocks inhibited cellulase catalyzed glucose release from L. digitata. Nevertheless, combined alginate lyase and cellulase treatment for 24 hours released all potential glucose regardless of the applied lyase. The enzymatic treatment moreover induced...

Hydrogen sulfide synthesis enzymes reduced in lower esophageal sphincter of patients with achalasia.

Science.gov (United States)

Zhang, L; Zhao, W; Zheng, Z; Wang, T; Zhao, C; Zhou, G; Jin, H; Wang, B

2016-10-01

The etiology of achalasia remains largely unknown. Considerable evidence reveals that the lower esophageal sphincter dysfunction is due to the lack of inhibitory neurotransmitter, secondary to esophageal neuronal inflammation or loss. Recent studies suggest hydrogen sulfide may act as an inhibitory transmitter in gastrointestinal tract, but study about hydrogen sulfide in human esophagus still lack. The aim of the study was to investigate if hydrogen sulfide synthesis enzymes could be detected in human esophagus and if the synthesis of the endogenous hydrogen sulfide could be affected in achalasia patients. Tissue samples in cardia, lower esophageal sphincter, 2 cm and 4 cm above lower esophageal sphincter were obtained from achalasia patients undergoing peroral endoscopic myotomy. Control tissues in lower esophageal sphincter were obtained from esophageal carcinoma patients. Expression of cystathionine-β-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients and control were detected by immunohistochemical staining. In addition, expression of cystathionine-β-synthase and cystathionine-γ-lyase were compared among different parts of esophagus in achalasia patients. Compared with control, the expression of cystathionine-β-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients was significantly reduced (χ 2 = 11.429, P = 0.010). The expression of cystathionine-β-synthase and cystathionine-γ-lyase were lower in lower esophageal sphincter than that in 2 cm and 4 cm above lower esophageal sphincter, respectively (all P achalasia, which implicates the involvement of the two hydrogen sulfide synthesis enzymes in the pathophysiology of achalasia. © 2015 International Society for Diseases of the Esophagus.

Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III

Science.gov (United States)

Aspinwall, Richard; Rothwell, Dominic G.; Roldan-Arjona, Teresa; Anselmino, Catherine; Ward, Christopher J.; Cheadle, Jeremy P.; Sampson, Julian R.; Lindahl, Tomas; Harris, Peter C.; Hickson, Ian D.

1997-01-01

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells. PMID:8990169

Enhancing RGI lyase thermostability by targeted single point mutations

DEFF Research Database (Denmark)

Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

2013-01-01

Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering...

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

Energy Technology Data Exchange (ETDEWEB)

Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena [Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (Italy); Indiveri, Cesare, E-mail: indiveri@unical.it [Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (Italy)

2012-05-25

Highlights: Black-Right-Pointing-Pointer mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. Black-Right-Pointing-Pointer The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. Black-Right-Pointing-Pointer The protein solubilised in Triton X-100 has been reconstituted in liposomes. Black-Right-Pointing-Pointer Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni{sup 2+}-chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [{sup 3}H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K{sub m} for carnitine was 36 {mu}M. [{sup 3}H]carnitine transport was inhibited by carnitine analogues and some Cys and NH{sub 2} reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

Structural Insights Into The Bacterial Carbon-Phosphorus Lyase Machinery

DEFF Research Database (Denmark)

Brodersen, Ditlev Egeskov

the proteins encoded in the phn operon act in concert to catabolise phosphonate remain unknown. We have determined the crystal structure of a 240 kDa Escherichia coli carbon-phosphorus lyase core complex at 1.7 Å and show that it comprises a highly intertwined network of subunits with several unexpected......Phosphonate compounds act as a nutrient source for some microorganisms when phosphate is limiting but require a specialised enzymatic machinery due to the presence of the highly stable carbon-phosphorus bond. Despite the fundamental importance to microbial metabolism, the details of how...... structural features. The complex contains at least two different active sites and suggest a revision of current models of carbon-phosphorus bond cleavage. Using electron microscopy, we map the binding site of an additional protein subunit, which may use ATP for driving conformational changes during...

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

OpenAIRE

Roberts, D P; Berman, P M; Allen, C; Stromberg, V K; Lacy, G H; Mount, M S

1986-01-01

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that en...

Elimination of hydrogen sulphide and β substitution in cystein, catalyzed by the cysteine-lyase of hens yolk-sac and yolk (1961)

International Nuclear Information System (INIS)

Chapeville, F.; Fromageot, P.

1961-01-01

The yolk of incubated hen's eggs contains a pyridoxal phosphate activated enzyme, free of iron, copper, magnesium and calcium. This enzyme activates the β-carbon atom of cysteine. Its reactivity is demonstrated by the ease with which this β-carbon fixes various sulfur containing substances in which the sulfur has reducing properties: inorganic sulfide, sulfide or cysteine itself. In the absence of substances able to react with the β-carbon atom, the active complex, consisting of the enzyme and the aminated tri-carbon chain, is hydrolysed to pyruvic acid and ammonia. The liberation of hydrogen sulfide thus appears to be the consequence either of the substitution of the β-carbon atom of cysteine or of the decomposition of the complex which this aminoacid forms with the enzyme studied. The latter seems therefore to possess an activity which differs from the activity of the desulfhydrases as yet known. We suggest to call this enzyme cystein-lyase. (authors) [fr

A review of machine learning methods to predict the solubility of overexpressed recombinant proteins in Escherichia coli.

Science.gov (United States)

Habibi, Narjeskhatoon; Mohd Hashim, Siti Z; Norouzi, Alireza; Samian, Mohammed Razip

2014-05-08

Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods. This paper presents an extensive review of the existing models to predict protein solubility in Escherichia coli recombinant protein overexpression system. The models are investigated and compared regarding the datasets used, features, feature selection methods, machine learning techniques and accuracy of prediction. A discussion on the models is provided at the end. This study aims to investigate extensively the machine learning based methods to predict recombinant protein solubility, so as to offer a general as well as a detailed understanding for researches in the field. Some of the models present acceptable prediction performances and convenient user interfaces. These models can be considered as valuable tools to predict recombinant protein overexpression results before performing real laboratory experiments, thus saving labour, time and cost.

Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

Science.gov (United States)

Ben-Daniel, Bat-Hen; Bar-Zvi, Dudy; Tsror Lahkim, Leah

2012-02-01

Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.

Science.gov (United States)

Yoshida, Akihiro; Yoshimura, Mamiko; Ohara, Naoya; Yoshimura, Shigeru; Nagashima, Shiori; Takehara, Tadamichi; Nakayama, Koji

2009-11-01

Hydrogen sulfide is one of the predominant volatile sulfur compounds (VSCs) produced by oral bacteria. This study developed and evaluated a system for detecting hydrogen sulfide production by oral bacteria. L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (METase) and beta carbon-sulfur (beta C-S) lyase were used to degrade homocysteine and cysteine, respectively, to produce hydrogen sulfide. Enzymatic reactions resulting in hydrogen sulfide production were assayed by reaction with bismuth trichloride, which forms a black precipitate when mixed with hydrogen sulfide. The enzymatic activities of various oral bacteria that result in hydrogen sulfide production and the capacity of bacteria from periodontal sites to form hydrogen sulfide in reaction mixtures containing L-cysteine or DL-homocysteine were assayed. With L-cysteine as the substrate, Streptococcus anginosus FW73 produced the most hydrogen sulfide, whereas Porphyromonas gingivalis American Type Culture Collection (ATCC) 33277 and W83 and Fusobacterium nucleatum ATCC 10953 produced approximately 35% of the amount produced by the P. gingivalis strains. Finally, the hydrogen sulfide found in subgingival plaque was analyzed. Using bismuth trichloride, the hydrogen sulfide produced by oral bacteria was visually detectable as a black precipitate. Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

Abundance and genetic diversity of microbial polygalacturonase and pectate lyase in the sheep rumen ecosystem.

Directory of Open Access Journals (Sweden)

Peng Yuan

Full Text Available Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen.A total of 103 unique fragments of polygalacturonase (PF00295 and pectate lyase (PF00544 and PF09492 genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65% with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles.This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions.

Regulation of expression of pectate lyase genes pelA, pelD, and pelE in Erwinia chrysanthemi.

Science.gov (United States)

Reverchon, S; Robert-Baudouy, J

1987-06-01

The regulation of pelA, pelD, and pelE genes encoding three of the five major pectate lyase isoenzymes (PLa, PLd, and PLe) in Erwinia chrysanthemi B374 was analyzed by using genetic fusions to lacZ. These three genes are clustered on a 5-kilobase DNA fragment in the order pelD-pelE-pelA and constitute three independent transcriptional units. We localized the pelDEA cluster near the pro-1 marker on the genetic map of B374 by chromosomal mobilization with RP4::mini-Mu plasmid pULB110. Three classes of regulatory mutations responsible for constitutive pectate lyase synthesis have been described (kdgR, gpiR, and cri). We studied the effects of each mutation on pelE, pelD, and pelA expression independently. The mutations kdgR and gpiR mainly affect the expression of pelE and pelD, although PLa synthesis is slightly increased. The cri mutation results in a low level of constitutive expression of the three pel genes, but it is a pleiotropic mutation since other genes not involved in pectinolysis are also affected. In addition, we demonstrated that exuR, a negative regulatory gene governing the catabolism of hexuronates, does not modify the expression of pel genes. The frequency of gpiR or cri mutations (about 10(-8)) and the resulting constitutivity of pectate lyase synthesis suggest that these genes act as negative regulatory genes in addition to kdgR, which is already known to encode a repressor. Moreover, we found that expression of pel-lac fusions carried on pBR322 derivatives was higher in E. chrysanthemi than in Escherichia coli; this fact suggests the existence of positive regulation of pectate lyase synthesis in E. chrysanthemi.

In Silico Characterization of Pectate Lyase Protein Sequences from Different Source Organisms

Directory of Open Access Journals (Sweden)

Amit Kumar Dubey

2010-01-01

Full Text Available A total of 121 protein sequences of pectate lyases were subjected to homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis. The phylogenetic tree constructed revealed different clusters based on different source organisms representing bacterial, fungal, plant, and nematode pectate lyases. The multiple accessions of bacterial, fungal, nematode, and plant pectate lyase protein sequences were placed closely revealing a sequence level similarity. The multiple sequence alignment of these pectate lyase protein sequences from different source organisms showed conserved regions at different stretches with maximum homology from amino acid residues 439–467, 715–816, and 829–910 which could be used for designing degenerate primers or probes specific for pectate lyases. The motif analysis revealed a conserved Pec_Lyase_C domain uniformly observed in all pectate lyases irrespective of variable sources suggesting its possible role in structural and enzymatic functions.

Evaluation of the hydroxynitrile lyase activity in cell cultures of capulin (Prunus serotina).

Science.gov (United States)

Hernández, Liliana; Luna, Héctor; Navarro-Ocaña, Arturo; Olivera-Flores, Ma Teresa de Jesús; Ayala, Ivon

2008-07-01

Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyase activity. The three week old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparations obtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding cyanohydrin (mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks, internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaldehyde with a modest to excellent enantioselectivity.

Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

International Nuclear Information System (INIS)

Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

2006-01-01

The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2 1 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

Directory of Open Access Journals (Sweden)

Siddiqui A.A.

2000-09-01

Full Text Available A full length cDNA (1463 bp encoding isocitrate lyase (EC 4.1.3.1 of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69 % A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.

Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314

Directory of Open Access Journals (Sweden)

Fei Wu

2016-03-01

Full Text Available A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB producing genes (hoxI and pcyA, while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB and the lyase gene (cpcU, cpcS, or cpcU/S were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.

The hydroxynitrile lyase from almond: crystal structure and mechanistical studies

International Nuclear Information System (INIS)

Dreveny, Ingrid

2001-09-01

Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase (HNL), a key enzyme of this process, cleaves a cyanohydrin precursor into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in industrial biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, α/β hydrolases and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. The high resolution crystal structure of the hydroxynitrile lyase from almond (Prunus amygdalus), PaHNL1, has been determined and constitutes the first 3D structure of an FAD-HNL. The overall fold and the architecture of the active site region showed that PaHNL1 belongs to the glucose-methanol-choline-oxidoreductase family, with closest structural similarity to glucose oxidase. There is strong evidence from the sequence and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Structures of PaHNL1 in complex with its natural substrate mandelonitrile and the competitive inhibitor benzyl alcohol provided insight into the residues involved in catalysis and a mechanism without participation of the cofactor could be suggested. Although the catalytic residues differ between the α/β-hydrolase-type HNLs and PaHNL1, common general features relevant for hydroxynitrile lyase activity could be proposed. (author)

Studies on pectin lyase

NARCIS (Netherlands)

Houdenhoven, van F.E.A.

1975-01-01

The pectin lyase activity in the commercial enzyme preparation Ultrazym originates from more then one type of enzyme; two of them, accounting for 95 % of the total activity, have been completely purified. As purity criteria specific activity, polyacrylamide disc gel electrophoresis and SDS

Improvement of daptomycin yield by overexpression of the ...

African Journals Online (AJOL)

The effects of the accessory genes flanking the non-ribosomal peptide synthetase (NRPS) genes on daptomycin production were investigated by overexpression under the control of ermE* promoter via the integrative Escherichia coli–Streptomyces vector pIB139. The yield of daptomycin was promoted significantly when ...

Hydrogen isotopes in individual amino acids reflect differentiated pools of hydrogen from food and water in Escherichia coli.

Science.gov (United States)

Fogel, Marilyn L; Griffin, Patrick L; Newsome, Seth D

2016-08-09

Hydrogen isotope (δ(2)H) analysis is widely used in animal ecology to study continental-scale movement because δ(2)H can trace precipitation and climate. To understand the biochemical underpinnings of how hydrogen is incorporated into biomolecules, we measured the δ(2)H of individual amino acids (AAs) in Escherichia coli cultured in glucose-based or complex tryptone-based media in waters with δ(2)H values ranging from -55‰ to +1,070‰. The δ(2)H values of AAs in tryptone spanned a range of ∼250‰. In E. coli grown on glucose, the range of δ(2)H among AAs was nearly 200‰. The relative distributions of δ(2)H of AAs were upheld in cultures grown in enriched waters. In E. coli grown on tryptone, the δ(2)H of nonessential AAs varied linearly with the δ(2)H of media water, whereas δ(2)H of essential AAs was nearly identical to δ(2)H in diet. Model calculations determined that as much as 46% of hydrogen in some nonessential AAs originated from water, whereas no more than 12% of hydrogen in essential AAs originated from water. These findings demonstrate that δ(2)H can route directly at the molecular level. We conclude that the patterns and distributions in δ(2)H of AAs are determined through biosynthetic reactions, suggesting that δ(2)H could become a new biosignature for studying novel microbial pathways. Our results also show that δ(2)H of AAs in an organism's tissues provides a dual tracer for food and environmental (e.g., drinking) water.

Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza 1

Science.gov (United States)

Gordon, William R.; Koukkari, Willard L.

1978-01-01

The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.” PMID:16660569

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

Science.gov (United States)

Choutko, Alexandra; Eichenberger, Andreas P; Gunsteren, Wilfred F; Dolenc, Jožica

2013-01-01

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. PMID:23595942

Exploration of swapping enzymatic function between two proteins: a simulation study of chorismate mutase and isochorismate pyruvate lyase.

Science.gov (United States)

Choutko, Alexandra; Eichenberger, Andreas P; van Gunsteren, Wilfred F; Dolenc, Jožica

2013-06-01

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. © 2013 The Protein Society.

Utilization of Aspergillus oryzae to produce pectin lyase from various agro-industrial residues

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Safia Koser

2014-07-01

Full Text Available The present study was aimed to investigate the culture influence on pectin lyase production potential of fungal strain Aspergillus oryzae. The enzyme profile of A. oryzae showed highest activity of pectin lyase after 3rd day of incubation on lemon peel waste under solid state fermentation conditions. To induce the pectin lyase synthesis capability of A. oryzae at optimal level various culture variables including physical and nutritional parameters were optimized by adopting classical optimization technique. Therefore, through fermentation process optimization the production of pectin lyase was substantially induced up to the level of 875 U/mL, when fermentation medium of lemon peel waste inoculated with 5 mL spore suspension of A. oryzae. The optimal fermentation conditions for maximum pectin lyase yield were as: optimum pH 5, 70% moisture level and incubated at 40 °C in addition with 1% sterile glucose solution as readily available carbon source and 0.2% yeast extract as an inexpensive nitrogen supplement (1%. The results obtained in current investigation so far demonstrated that culture conditions have great influence on the pectin lyase production potential of A. oryzae.

Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

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Steven M Swift

Full Text Available Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

Structure and Mechanism of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway

DEFF Research Database (Denmark)

He, Shu-Mei; Wathier, Matthew; Podzelinska, Kateryna

2011-01-01

PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-β-lactamase s......PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo...

Prenatal diagnosis in adenylosuccinate lyase deficiency

NARCIS (Netherlands)

Marie, S.; Flipsen, J. W.; Duran, M.; Poll-The, B. T.; Beemer, F. A.; Bosschaart, A. N.; Vincent, M. F.; van den Berghe, G.

2000-01-01

Adenylosuccinate lyase deficiency, an autosomal recessive inborn error of purine synthesis, provokes accumulation in body fluids of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Most patients display severe

Active tuberculosis patients have high levels of IgA anti-alpha-crystallin and isocitrate lyase proteins.

Science.gov (United States)

Talavera-Paulín, M; García-Morales, L; Ruíz-Sánchez, B P; Caamal-Ley, Á D; Hernández-Solis, A; Ramírez-Casanova, E; Cicero-Sabido, R; Espitia, C; Helguera-Repetto, C; González-Y-Merchand, J A; Flores-Mejía, R; Estrada-Parra, S; Estrada-García, I; Chacón-Salinas, R; Wong-Baeza, I; Serafín-López, J

2016-12-01

Mexico City, Mexico. To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

Production and Purification of a Novel Xanthan Lyase from a Xanthan-Degrading Microbacterium sp. Strain XT11

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Fan Yang

2014-01-01

Full Text Available A xanthan lyase was produced and purified from the culture supernatant of an excellent xanthan-modifying strain Microbacterium sp. XT11. Xanthan lyase was induced by xanthan but was inhibited by its structural monomer glucose. Its production by strain XT11 is much higher than that by all other reported strains. The purified xanthan lyase has a molecular mass of 110 kDa and a specific activity of 28.2 U/mg that was much higher than that of both Paenibacillus and Bacillus lyases. It was specific on the pyruvated mannosyl residue in the intact xanthan molecule, but about 50% lyase activity remained when xanthan was partially depyruvated. Xanthan lyase was optimally active at pH 6.0–6.5 and 40°C and alkali-tolerant at a high pH value of 11.0. The metal ions including K+, Ca2+, Na+, Mg2+, Mn2+, and Li+ strongly stimulated xanthan lyase activity but ions Zn2+ and Cu2+ were its inhibitor. Xanthan lyase should be a novel enzyme different from the other xanthan lyases ever reported.

Utilization of Aspergillus oryzae to produce pectin lyase from various agro-industrial residues

OpenAIRE

Koser, Safia; Anwar, Zahid; Iqbal, Zafar; Anjum, Awais; Aqil, Tahir; Mehmood, Sajid; Irshad, Muhammad

2014-01-01

The present study was aimed to investigate the culture influence on pectin lyase production potential of fungal strain Aspergillus oryzae. The enzyme profile of A. oryzae showed highest activity of pectin lyase after 3rd day of incubation on lemon peel waste under solid state fermentation conditions. To induce the pectin lyase synthesis capability of A. oryzae at optimal level various culture variables including physical and nutritional parameters were optimized by adopting classical optimiza...

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

Science.gov (United States)

Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

2005-01-01

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange

DEFF Research Database (Denmark)

Rist, Wolfgang; Jørgensen, Thomas J D; Roepstorff, Peter

2003-01-01

Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock...... gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37...... degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock...

Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli.

Science.gov (United States)

Deng, Yu; Ma, Ning; Zhu, Kangjia; Mao, Yin; Wei, Xuetuan; Zhao, Yunying

2018-03-01

The glyoxylate shunt is a branch of the tricarboxylic acid (TCA) cycle which directly determines the synthesis of glycolate, and the balance between the glyoxylate shunt and TCA cycle is very important for the growth of Escherichia coli. In order to accumulate glycolate at high yield and titer, strategies for over-expressing glycolate pathway enzymes including isocitrate lyase (AceA), isocitrate dehydrogenase kinase/phosphatase (AceK) and glyoxylate reductase (YcdW) were analyzed. The genes encoding these three enzymes were transcribed under the control of promoter pTrc on pTrc99A, to form pJNU-3, which was harbored by strain Mgly1, resulting in strain Mgly13. Strain Mgly13 produced glycolate with 0.385 g/g-glucose yield (45.2% of the theoretical yield). Citrate synthase (GltA) converted excess acetyl-CoA and oxaloacetate to citrate and was over-expressed by pJNU-4 (pCDFDuet-1 backbone). Thus, the resulting strain Mgly134 produced glycolate with a 0.504 g/g-glucose yield (59.3% of the theoretical yield). We then eliminated the pathways involved in the degradation of glycolate, resulting in strain Mgly434, which produced glycolate with 92.9% of the theoretical yield. Following optimization of fermentation, the maximum glycolate titer from strain Mgly434 was 65.5 g/L. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1

NARCIS (Netherlands)

Ruijssenaars, H.J.; Hartmans, S.; Verdoes, J.C.

2000-01-01

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding

Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

Science.gov (United States)

Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteolytic activity of Escherichia coli oligopeptidase B against proline-rich antimicrobial peptides

DEFF Research Database (Denmark)

Mattiuzzo, Maura; De Gobba, Cristian; Runti, Giulia

2014-01-01

Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes...

Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

Science.gov (United States)

Guerra, D.; Anderson, A. J.; Salisbury, F. B.

1985-01-01

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

Chondroitin Sulfate (CS) Lyases: Structure, Function and Application in Therapeutics.

Science.gov (United States)

Rani, Aruna; Patel, Seema; Goyal, Arun

2018-01-01

Glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) are the chief natural polysaccharides which reside in biological tissues mainly in extracellular matrix. These CS along with adhesion molecules and growth factors are involved in central nervous system (CNS) development, cell progression and pathogenesis. The chondroitin lyases are the enzyme that degrade and alter the CS chains and hence modify various signalling pathways involving CS chains. These CS lyases are substrate specific, can precisely manipulate the CS polysaccharides and have various biotechnological, medical and therapeutic applications. These enzymes can be used to produce the unsaturated oligosaccharides, which have immune-modulatory, anti-inflammatory and neuroprotective properties. This review focuses on the major breakthrough of the chondroitin sulfate degrading enzymes, their structures and functioning mechanism. This also provides comprehensive information regarding production, purification, characterization of CS lyases and their major applications, both established as well as emerging ones such as neural development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cloning and over-expression of Penicillin G acylase in Escherichia ...

African Journals Online (AJOL)

The aim of this study is to screen for PGA producing Escherichia coli isolates as well as the cloning and recombinant expression of PGA for high level enzyme production. Bacteria isolated from environmental and clinical samples were identified by standard microbiological tests and then E. coli isolates were subjected to

[Construction and high-density fermentation of alkaline pectate lyase high-yield yeast].

Science.gov (United States)

Wang, Xiaowen; Xiang, La; Xu, Ting; Lu, Zhenghui; Zhang, Guimin

2017-12-25

Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.

Comparison of expression, purification and characterization of a new pectate lyase from Phytophthora capsici using two different methods

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Zhang Xiuguo

2011-04-01

Full Text Available Abstract Background Pectate lyases (PELs play an important role in the infection process of plant pathogens and also have a commercial significance in industrial applications. Most of the PELs were expressed as soluble recombinant proteins, while a few recombinant proteins were insoluble. The production of a large-scale soluble recombinant PEL would allow not only a more detailed structural and functional characterization of this enzyme but also may have important applications in the food industry. Results We cloned a new pectate lyase gene (Pcpel2 from Phytophthora capsici. Pcpel2 was constructed by pET system and pMAL system, and both constructs were used to express the PCPEL2 in Escherichia coli BL21 (DE3 pLysS. The expressed products were purified using affinity chromatography and gel filtration chromatography. The purity, specific activity and pathogenicity of the purified PCPEL2 expressed by the pMAL system were higher than the purified PCPEL2 expressed by the pET system. In addition, some other characteristics of the purified PCPEL2 differed from the two systems, such as crystallographic features. Purified PCPEL2 expressed by the pMAL system was crystallized by the hanging-drop vapour-diffusion method at 289 K, and initial crystals were grown. Conclusion The two different methods and comparison presented here would be highly valuable in obtaining an ideal enzyme for the downstream experiments, and supply an useful alternative to purify some insoluble recombinant proteins.

Bio-hydrogen production by Enterobacter asburiae SNU-1 isolated from a landfill

Energy Technology Data Exchange (ETDEWEB)

Jong-Hwan Shin; Jong Hyun Yoon; Tai Hyun Park [School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, (Korea, Republic of)

2006-07-01

A new fermentative hydrogen-producing bacterium was isolated from a landfill, and it was identified as Enterobacter asburiae strain using a genomic DNA hybridization method. Environmental factors and metabolic flux influencing the hydrogen production were investigated, including pH, initial glucose and formate concentrations. The major hydrogen production pathway of this strain is considered to be a formate pathway by using formate hydrogen lyase (FHL). Optimum pH for the hydrogen production was pH 7.0 in PYG medium, at which hydrogen production/unit volume and overall hydrogen productivity were 2615 ml/l and 174 ml H{sub 2}/l/hr, respectively, at 25 g glucose/l. The maximum hydrogen productivity was estimated to be 417 ml H{sub 2}/l/hr at 15 g glucose/l. This strain produced bio-hydrogen mostly in the stationary phase, in which formate concentration was high. In this paper, hydrogen production was tried in formate medium after cell harvest. (authors)

Bio-hydrogen production by Enterobacter asburiae SNU-1 isolated from a landfill

International Nuclear Information System (INIS)

Jong-Hwan Shin; Jong Hyun Yoon; Tai Hyun Park

2006-01-01

A new fermentative hydrogen-producing bacterium was isolated from a landfill, and it was identified as Enterobacter asburiae strain using a genomic DNA hybridization method. Environmental factors and metabolic flux influencing the hydrogen production were investigated, including pH, initial glucose and formate concentrations. The major hydrogen production pathway of this strain is considered to be a formate pathway by using formate hydrogen lyase (FHL). Optimum pH for the hydrogen production was pH 7.0 in PYG medium, at which hydrogen production/unit volume and overall hydrogen productivity were 2615 ml/l and 174 ml H 2 /l/hr, respectively, at 25 g glucose/l. The maximum hydrogen productivity was estimated to be 417 ml H 2 /l/hr at 15 g glucose/l. This strain produced bio-hydrogen mostly in the stationary phase, in which formate concentration was high. In this paper, hydrogen production was tried in formate medium after cell harvest. (authors)

Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

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Agnes Csanadi

Full Text Available Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids. In cancer, Malic enzyme (ME and ATP-citrate lyase (ACLY are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC.ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival.Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.

Characterization of a Novel Alginate Lyase from Marine Bacterium Vibrio furnissii H1

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Xiaoyan Zhu

2018-01-01

Full Text Available Alginate lyases show great potential for industrial and medicinal applications, especially as an attractive biocatalyst for the production of oligosaccharides with special bioactivities. A novel alginate lyase, AlyH1, from the marine bacterium Vibrio furnissii H1, which has been newly isolated from rotten seaweed, was purified and characterized. The purified enzyme showed the specific activity of 2.40 U/mg. Its molecular mass was 35.8 kDa. The optimal temperature and pH were 40 °C and pH 7.5, respectively. AlyH1 maintained stability at neutral pH (7.0–8.0 and temperatures below 30 °C. Metal ions Na+, Mg2+, and K+ increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax values of AlyH1 were 2.28 mg/mL and 2.81 U/mg, respectively. AlyH1 exhibited activities towards both polyguluronate and polymannuronate, and preferentially degraded polyguluronate. Products prepared from sodium alginate by AlyH1 were displayed to be di-, tri-, and tetra-alginate oligosaccharides. A partial amino acid sequence (190 aa of AlyH1 analysis suggested that AlyH1 was an alginate lyase of polysaccharide lyase family 7. The sequence showed less than 77% identity to the reported alginate lyases. These data demonstrated that AlyH1 could be as a novel and potential candidate in application of alginate oligosaccharides production with low polymerization degrees.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

Science.gov (United States)

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis

OpenAIRE

Yamamoto, Sayo; Sahara, Takehiko; Sato, Daisuke; Kawasaki, Kosei; Ohgiya, Satoru; Inoue, Akira; Ojima, Takao

2008-01-01

Alginate lyase is an enzyme that degrades alginate chains via β-elimination and has been used for the production of alginate oligosaccharides and protoplasts from brown algae. Previously, we deduced the amino-acid sequence of an abalone alginate lyase, HdAly, from its cDNA sequence and, through multiple amino-acid sequence alignment, found that several basic amino-acid residues were highly conserved among the polysaccharide-lyase family 14 (PL-14) enzymes including HdAly. In the present study...

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

DEFF Research Database (Denmark)

da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

2014-01-01

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

A radiometric technique for the measurement of adenylosuccinate lyase

International Nuclear Information System (INIS)

Park, K.W.; Tyagi, A.K.; Cooney, D.A.

1980-01-01

When radioactive adenylosuccinic acid (AMP-S) is metabolized to AMP and fumaric acid by the enzyme adenylosuccinate lyase (EC 4.3.2.2), a proton is released to the solvent as 3 H 2 O. This removal is believed to be stereospecifically identical to that catalyzed by the enzyme, L-aspartase, and therefore entails the loss of a proton from C-3 of the dicarboxylic acid moiety of the nucleotide. Advantage has been taken of this fact in the design of a facile assay for this enzyme. The assay permits the simultaneous estimation of the lyase activity in a large battery of samples; it is not interfered with by opalescent or proteinaceous suspensions; it is accurate and outstandingly sensitive. (Auth.)

Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Noordermeer, M.A.; Veldink, G.A.

1999-01-01

Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been

Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

Science.gov (United States)

Minerdi, Daniela; Zgrablic, Ivan; Castrignanò, Silvia; Catucci, Gianluca; Medana, Claudio; Terlizzi, Maria Elena; Gribaudo, Giorgio; Gilardi, Gianfranco; Sadeghi, Sheila J

2016-01-01

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

NARCIS (Netherlands)

Bot, Martine; Van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; Van Santbrink, Peter J.; Westra, Marijke M.; Van Der Hoeven, Gerd; Gijbels, Marion J.; Mueller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; Van Berkel, Theo J. C.; Nofer, Jerzy-Roch; Bot, Ilze; Biessen, Erik A. L.

2013-01-01

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets

Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice

NARCIS (Netherlands)

Bot, Martine; van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; van Santbrink, Peter J.; Westra, Marijke M.; van der Hoeven, Gerd; Gijbels, Marion J.; Müller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; van Berkel, Theo J. C.; Nofer, Jerzy-Roch; Bot, Ilze; Biessen, Erik A. L.

2013-01-01

Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to

Role of overexpressed CFA/I fimbriae in bacterial swimming.

Science.gov (United States)

Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

2012-06-01

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 CFA/I fimbriae on bacterial swimming motility.

Hydrogen production by Escherichia coli {delta}hycA {delta}lacI using cheese whey as substrate

Energy Technology Data Exchange (ETDEWEB)

Rosales-Colunga, Luis Manuel; Ordonez, Leandro G.; De Leon-Rodriguez, Antonio (Division de Biologia Molecular, Instituto Potosino de Investigacion Cientifica y Tecnologica, Camino a la Presa San Jose 2055, Col. Lomas 4a secc. CP 78216, San Luis Potosi, SLP. Mexico); Razo-Flores, Elias; Alatriste-Mondragon, Felipe (Division de Ciencias Ambientales, Instituto Potosino de Investigacion Cientifica y Tecnologica, Camino a la Presa San Jose 2055, Col. Lomas 4a secc. CP 78216, San Luis Potosi, SLP. Mexico)

2010-01-15

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli {delta}hycA {delta}lacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H{sub 2}/optical density at 600 nm (OD{sub 600nm}) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H{sub 2}/OD{sub 600nm} unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H{sub 2}/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures. (author)

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

NARCIS (Netherlands)

Bot, M.; Veldhoven, van P.P.; Jager, de S.C.; Johnson, J.; Nijstad, N.; van, Santbrink P.J.; Westra, M.M.; Hoeven, van der G.; Gijbels, M.J.; Muller-Tidow, C.; Varga, G.; Tietge, U.J.; Kuiper, J.; Berkel, van T.J.; Nofer, J.R.; Bot, I.; Biessen, E.A.

2013-01-01

Abstract Aims Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte

Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2

Energy Technology Data Exchange (ETDEWEB)

Lee, Su In; Kim, Hee Sook [Kyungsung Univ., Busan (Korea, Republic of). Dept. of Food Science and Biotechnology; Choi, Sung Hee; Lee, Eun Yeol [Kyung Hee Univ., Gyeonggi-do (Korea, Republic of). Dept. of Chemical Engineering

2012-09-15

A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate. (orig.)

Overexpression of a eukaryotic glutathione reductase gene from Brassica campestris improved resistance to oxidative stress in Escherichia coli

International Nuclear Information System (INIS)

Yoon, Ho-Sung; Lee, In-Ae; Lee, Hyoshin; Lee, Byung-Hyun; Jo, Jinki

2005-01-01

Glutathione reductase (GR) plays an essential role in a cell's defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant glutathione. We constructed a recombinant plasmid based on the expression vector pET-18a that overexpresses a eukaryotic GR from Brassica campestris (BcGR) in Escherichia coli. For comparative analyses, E. coli GR (EcGR) was also subcloned in the same manner. The transformed E. coli with the recombinant constructs accumulated a high level of GR transcripts upon IPTG induction. Also, Western blot analysis showed overproduction of the BcGR protein in a soluble fraction of the transformed E. coli extract. When treated with oxidative stress generating reagents such as paraquat, salicylic acid, and cadmium, the BcGR overproducing E. coli exhibited a higher level of growth and survival rate than the control E. coli strain, but it was not as high as the E. coli strain transformed with the inducible EcGR. The translated amino acid sequences of BcGR and EcGR share 37.3% identity but all the functionally known important residues are conserved. It appears that eukaryotic BcGR functions in a prokaryotic system by providing protection against oxidative damages in E. coli

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

DEFF Research Database (Denmark)

Kruse, Thomas; Møller-Jensen, Jakob; Løbner-Olesen, Anders

2003-01-01

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow...... cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell...... that MreB filaments participate in directional chromosome movement and segregation....

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

Science.gov (United States)

Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

2015-06-01

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

Photosensitized production of hydrogen by Halobacterium halobium MMT sub 22 coupled to Escherichia coli in reversed micelles of sodium lauryl sulfate in organic solvents

Energy Technology Data Exchange (ETDEWEB)

Khan, M.M.T.; Bhatt, J.P. (Central Salt and Marine Research Inst., Bhavnagar (India))

1991-01-01

Observation on the enhanced production of hydrogen by Halobacterium halobium MMT{sub 22} coupled to Escherichia coli entrapped inside the reversed micelles formed by sodium lauryl sulfate in various organic solvents, namely benzene, carbon tetrachloride, toluene, n-heptane, nitrobenzene, chlorobenzene, are reported. In the present system, a hundred fold increase in activity as compared to the activity in the usual aqueous medium was observed. (author).

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

Science.gov (United States)

Barbosa, Marina R; Sampaio, Igor H; Teodoro, Bruno G; Sousa, Thais A; Zoppi, Claudio C; Queiroz, André L; Passos, Madla A; Alberici, Luciane C; Teixeira, Felipe R; Manfiolli, Adriana O; Batista, Thiago M; Cappelli, Ana Paula Gameiro; Reis, Rosana I; Frasson, Danúbia; Kettelhut, Isis C; Parreiras-e-Silva, Lucas T; Costa-Neto, Claudio M; Carneiro, Everardo M; Curi, Rui; Silveira, Leonardo R

2013-10-01

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids. Copyright © 2013 Elsevier B.V. All rights reserved.

Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids.

Science.gov (United States)

Turner, Nicholas J

2011-04-01

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison of hydrogen-production capability of four different Enterobacteriaceae strains under growing and non-growing conditions

Energy Technology Data Exchange (ETDEWEB)

Seol, Eunhee; Kim, Seohyoung; Raj, S. Mohan; Park, Sunghoon [Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735 (Korea)

2008-10-15

Non-growing cells can function as whole-cell biocatalysts for hydrogen (H{sub 2}) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H{sub 2}-production capability of four Enterobacteriaceae strains (Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H{sub 2}-production activity at varying temperatures (25-45 C) and pH conditions (6.0-8.0) using glucose or formate as the carbon source. Under growing conditions with 10 mM glucose as a substrate, E. aerogenes exhibited the highest H{sub 2}-production activity (17.0 {+-} 0.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}) among the four strains, but the final H{sub 2} yield was similar (1.7-1.8 mol H{sub 2} mol{sup -1} glucose) in all four strains. H{sub 2} production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20 mM formate as a substrate, we obtained high H{sub 2}-production activities, in the range of 95.5-195.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}, with E. coli DJT135 exhibiting the highest activity (195.2 {mu}mol H{sub 2} mg{sup -1} h{sup -1}) at pH 6.0 and 45 C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H{sub 2}-production activity to 6.1-7.7 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}. This study indicated that formate is a better substrate than glucose for H{sub 2} production by non-growing cells, and that the H{sub 2}-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655. (author)

Kinetic and thermodynamic properties of alginate lyase and cellulase co-produced by Exiguobacterium species Alg-S5.

Science.gov (United States)

Mohapatra, Bidyut R

2017-05-01

In an effort to screen out the alginolytic and cellulolytic bacteria from the putrefying invasive seaweed Sargassum species accumulated off Barbados' coast, a potent bacterial strain was isolated. This bacterium, which simultaneously produced alginate lyase and cellulase, was identified as Exiguobacterium sp. Alg-S5 via the phylogenetic approach targeting the 16S rRNA gene. The co-produced alginate lyase and cellulase exhibited maximal enzymatic activity at pH 7.5 and at 40°C and 45°C, respectively. The K m and V max values recorded as 0.91mg/mL and 21.8U/mg-protein, respectively, for alginate lyase, and 10.9mg/mL and 74.6U/mg-protein, respectively, for cellulase. First order kinetic analysis of the thermal denaturation of the co-produced alginate lyase and cellulase in the temperature range from 40°C to 55°C revealed that both the enzymes were thermodynamically efficient by displaying higher activation energy and enthalpy of denaturation. These enzymatic properties indicate the potential industrial importance of this bacterium in algal biomass conversion. This appears to be the first report on assessing the efficacy of a bacterium for the co-production of alginate lyase and cellulase. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

Energy Technology Data Exchange (ETDEWEB)

Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

2015-10-23

L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

Cystathionine .gamma.-lyase: Clinical, metabolic, genetic, and structural studies

Czech Academy of Sciences Publication Activity Database

Kraus, J. P.; Hašek, Jindřich; Kožich, V.; Collard, R.; Venezia, S.; Janošíková, B.; Wang, J.; Stabler, S. P.; Allen, R. H.; Jakobs, C.; Finn, C. T.; Chien, Y. H.; Hwu, W. L.; Hegele, R. A.; Mudd, S. H.

2009-01-01

Roč. 97, č. 4 (2009), s. 250-259 ISSN 1096-7192 R&D Projects: GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : cystathionine gamma-lyase * cystathioninuria * hypercystathioninemia Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.897, year: 2009

Phenylalanine ammonia-lyase (PAL) gene activity in response to ...

African Journals Online (AJOL)

Phenylalanine ammonia-lyase (PAL) catalyzes the biosynthesis of rosmarinic acid (RA), tyrosine and phenylalanine are the precursors of RA, while proline drives metabolite precursors toward Shikimate and phenylpropanoid pathway ending with the production of RA. The aim of this study was to investigate the PAL gene ...

Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

DEFF Research Database (Denmark)

2016-01-01

The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high...... substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well...

Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice.

Directory of Open Access Journals (Sweden)

Martine Bot

Full Text Available AIMS: Altered sphingosine 1-phosphate (S1P homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/- deficiency on leukocyte subsets relevant to atherosclerosis. METHODS AND RESULTS: LDL receptor deficient mice that were transplanted with Sgpl1(-/- bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/- chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. CONCLUSIONS: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

Science.gov (United States)

Allegrini, Alessandra; Astegno, Alessandra; La Verde, Valentina; Dominici, Paola

2017-04-01

Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

Science.gov (United States)

Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

2015-01-01

Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

Ammonia production from amino acid-based biomass-like sources by engineered Escherichia coli.

Science.gov (United States)

Mikami, Yosuke; Yoneda, Hisanari; Tatsukami, Yohei; Aoki, Wataru; Ueda, Mitsuyoshi

2017-12-01

The demand for ammonia is expected to increase in the future because of its importance in agriculture, industry, and hydrogen transportation. Although the Haber-Bosch process is known as an effective way to produce ammonia, the process is energy-intensive. Thus, an environmentally friendly ammonia production process is desired. In this study, we aimed to produce ammonia from amino acids and amino acid-based biomass-like resources by modifying the metabolism of Escherichia coli. By engineering metabolic flux to promote ammonia production using the overexpression of the ketoisovalerate decarboxylase gene (kivd), derived from Lactococcus lactis, ammonia production from amino acids was 351 mg/L (36.6% yield). Furthermore, we deleted the glnA gene, responsible for ammonia assimilation. Using yeast extract as the sole source of carbon and nitrogen, the resultant strain produced 458 mg/L of ammonia (47.8% yield) from an amino acid-based biomass-like material. The ammonia production yields obtained are the highest reported to date. This study suggests that it will be possible to produce ammonia from waste biomass in an environmentally friendly process.

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

Directory of Open Access Journals (Sweden)

Zou Ruiyang

2011-04-01

Full Text Available Abstract Background Accurate interpretation of quantitative PCR (qPCR data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism.

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Huan Peng

Full Text Available Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases. In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7. A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7 were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2 and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi. Similarly, this procedure reduced the number of female adults at 40 dpi

Cystathionine γ-lyase

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Halina Jurkowska

2014-01-01

Full Text Available γ-Cystathionase (CTH, EC: 4.4.1.1, an enzyme widely distributed in the world of prokaryotic and eukaryotic organisms, catalyzes the formation and transformations of sulfane sulfur-containing compounds and plays a pivotal role in the L-cysteine desulfuration pathway. Human, tetrameric CTH is composed of two dimers and each monomer binds pyridoxal phosphate (PLP. The gene, located on the short arm of chromosome 1, consists of 13 exons and 12 introns. As a result of alternative splicing, three isoforms of human CTH arise. Analysis of genetic variations of the CTH encoding gene showed a large number of polymorphisms. A decrease of the expression of CTH entails a drop in the level of cysteine , glutathione (GSH, taurine and hydrogen sulfide (H2S in the cells and, more importantly, leads to cystathioninuria. H2S, endogenously formed by CTH, affects the vasodilation and regulation of blood pressure. CTH knockout mice have decreased levels of H2S, hypertension, and reduced capacity for vascular endothelium relaxation. Overexpression of the gene encoding CTH in the cells leads to increased production of H2S. H2S plays a role in protection of neurons against oxidative stress, and stimulates an increase in γ-glutamylcysteine synthetase and thereby an increase in the level of GSH. Sulfurtransferases, including CTH, can locally prevent oxidative stress due to reversible oxidation of – SH groups in the presence of increased levels of reactive oxygen species, and reduction in the presence of GSH and/or reduced thioredoxin.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications

NARCIS (Netherlands)

Heberling, Matthew M.; Wu, Bian; Bartsch, Sebastian; Janssen, Dick B.

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the

One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

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Parinaz Ghadam

2017-05-01

Full Text Available Objective(s: Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

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He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-02-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator

International Nuclear Information System (INIS)

Sanctis, Daniele de; Rêgo, Ana T.; Marçal, David; McVey, Colin E.; Carrondo, Maria A.; Enguita, Francisco J.

2007-01-01

The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å. The sorbitol operon regulator (SorC) regulates the metabolism of l-sorbose in Klebsiella pneumonia. SorC was overexpressed in Escherichia coli and purified, and crystals were obtained of a tetrameric form. A single crystal showed X-ray diffraction to 3.20 Å. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 91.6, b = 113.3, c = 184.1 Å. Analysis of the molecular-replacement solution indicates the presence of four SorC molecules in the asymmetric unit

Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli

DEFF Research Database (Denmark)

Holm, Anders Koefoed; Blank, L.M.; Oldiges, M.

2010-01-01

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli...... of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli....

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

Science.gov (United States)

Plaga, W; Frank, R; Knappe, J

1988-12-15

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

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Adam E Brummett

Full Text Available The osmolyte dimethylsulfoniopropionate (DMSP is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS, a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121. Measurements of metal binding affinity and catalytic activity indicate that Fe(II is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II per monomer. Electronic absorption and electron paramagnetic resonance (EPR studies show an interaction between NO and Fe(II-DddW, with NO binding to the EPR silent Fe(II site giving rise to an EPR active species (g = 4.29, 3.95, 2.00. The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

DEFF Research Database (Denmark)

Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

2005-01-01

Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

Engineering Escherichia coli for improved ethanol production from gluconate.

Science.gov (United States)

Hildebrand, Amanda; Schlacta, Theresa; Warmack, Rebeccah; Kasuga, Takao; Fan, Zhiliang

2013-10-10

We report on engineering Escherichia coli to produce ethanol at high yield from gluconic acid (gluconate). Knocking out genes encoding for the competing pathways (l-lactate dehydrogenase and pyruvate formate lyase A) in E. coli KO11 eliminated lactate production, lowered the carbon flow toward acetate production, and improved the ethanol yield from 87.5% to 97.5% of the theoretical maximum, while the growth rate of the mutant strain was about 70% of the wild type. The corresponding genetic modifications led to a small improvement of ethanol yield from 101.5% to 106.0% on glucose. Deletion of the pyruvate dehydrogenase gene (pdh) alone improved the ethanol yield from 87.5% to 90.4% when gluconate was a substrate. The growth rate of the mutant strain was identical to that of the wild type. The corresponding genetic modification led to no improvements on ethanol yield on glucose. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of overexpressed CFA/I fimbriae in bacterial swimming

International Nuclear Information System (INIS)

Cao, Ling; Lim, Timothy; Jun, SangMu; Riccardi, Carol; Yang, Xinghong; Suo, Zhiyong; Deliorman, Muhammedin; Kellerman, Laura; Avci, Recep

2012-01-01

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility. (paper)

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

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Khanna, Namita; Ghosh, Ananta Kumar; Huntemann, Marcel; Deshpande, Shweta; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Pagani, Ioanna; Pati, Amrita; Pitluck, Sam; Nolan, Matt; Woyke, Tanja; Teshima, Hazuki; Chertkov, Olga; Daligault, Hajnalka; Davenport, Karen; Gu, Wei; Munk, Christine; Zhang, Xiaojing; Bruce, David; Detter, Chris; Xu, Yan; Quintana, Beverly; Reitenga, Krista; Kunde, Yulia; Green, Lance; Erkkila, Tracy; Han, Cliff; Brambilla, Evelyne-Marie; Lang, Elke; Klenk, Hans-Peter; Goodwin, Lynne; Chain, Patrick; Das, Debabrata

2013-12-20

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

Microbial β-etherases and glutathione lyases for lignin valorisation in biorefineries: current state and future perspectives.

Science.gov (United States)

Husarcíková, Jana; Voß, Hauke; Domínguez de María, Pablo; Schallmey, Anett

2018-05-04

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, β-etherases offer the possibility for a selective cleavage of the β-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. β-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on β-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Jendresen, Christian Bille; Stahlhut, Steen Gustav; Li, Mingji

2015-01-01

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches...

Pectinolytic bacteria and their secreted pectate lyases: agents for the maceration and solubilization of phytomass for fuels production

Energy Technology Data Exchange (ETDEWEB)

Preston, J.F. III; Rice, J.D.; Chow, M.C. (Florida Univ., Gainesville, FL (United States). Dept. of Microbiology and Cell Science)

1993-01-01

The objectives of this research have been to identify the pectinolytic enzymes secreted by bacteria and apply these towards the enhanced maceration and solubilization of plant material, focusing on the pectate lyases secreted by the phytopathogenic strains of Erwinia chrysanthemi, the ruminant resident Lachnospira multiparus, and the wood digestor isolate, Clostridium populeti. An HPLC approach has been developed that permits the kinetic analysis of each enzyme with respect to the formation of individual products during the pectate depolymerization process. This approach has demonstrated that each of these organisms secretes a nonrandom trimer-generating pectate lyase with a combination of endolytic and exolytic depolymerizing mechanisms. Two different strains of E. chrysanthemi secrete a battery of pectate lyases that include random endolytic as well as nonrandom dimer - and nonrandom trimer-generating endolytic/exolytic mechanisms. (author)

Catalytic Mechanisms and Biocatalytic Applications of Aspartate and Methylaspartate Ammonia Lyases

NARCIS (Netherlands)

de Villiers, Marianne; Veetil, Vinod Puthan; Raj, Hans; de Villiers, Jandre; Poelarends, Gerrit J.

2012-01-01

Ammonia lyases catalyze the formation of alpha-beta-unsaturated bonds by the elimination of ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts.

Science.gov (United States)

Guo, Yin; Bandaru, Viswanath; Jaruga, Pawel; Zhao, Xiaobei; Burrows, Cynthia J; Iwai, Shigenori; Dizdaroglu, Miral; Bond, Jeffrey P; Wallace, Susan S

2010-02-04

The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from gamma-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products

Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: escherichia coli tryptophan indole-lyase

International Nuclear Information System (INIS)

Kiick, D.M.; Phillips, R.S.

1988-01-01

Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1.

Science.gov (United States)

Maeda, Yasuhiro; Yagi, Hideki; Takemoto, Kana; Utsumi, Hiroyuki; Fukunari, Atsushi; Sugahara, Kunio; Masuko, Takashi; Chiba, Kenji

2014-05-01

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

Directory of Open Access Journals (Sweden)

Minussi Rosana Cristina

1998-01-01

Full Text Available The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL and polygalacturonase (PG by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3 in a rotary shaker (150 rpm for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium promoted higher PL activity and mycelial dry weight when compared to pectin and the use of sugar-cane juice and yeast extract yielded levels of PG activity that were similar to those obtained with sucrose-yeast extract or pectin. The results indicated that, even at low concentrations, sugar-cane juice was capable of inducing pectin lyase and polygalacturonase with no cellulase activity in P. griseoroseum.

Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

NARCIS (Netherlands)

Raj, Hans; Szymanski, Wiktor; de Villiers, Jandre; Rozeboom, Henriëtte J.; Puthan Veetil, Vinod; Reis, Carlos R.; Villiers, Marianne de; Dekker, Frank J.; Wildeman, Stefaan de; Quax, Wim J.; Thunnissen, Andy-Mark W.H.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source.

Science.gov (United States)

Valle, Antonio; Cabrera, Gema; Muhamadali, Howbeer; Trivedi, Drupad K; Ratray, Nicholas J W; Goodacre, Royston; Cantero, Domingo; Bolivar, Jorge

2015-09-01

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed-stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol-based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4-transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

NARCIS (Netherlands)

Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

2005-01-01

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation

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Danielle Biscaro Pedrolli

2014-01-01

Full Text Available A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

Ensemble modeling for aromatic production in Escherichia coli.

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Matthew L Rizk

2009-09-01

Full Text Available Ensemble Modeling (EM is a recently developed method for metabolic modeling, particularly for utilizing the effect of enzyme tuning data on the production of a specific compound to refine the model. This approach is used here to investigate the production of aromatic products in Escherichia coli. Instead of using dynamic metabolite data to fit a model, the EM approach uses phenotypic data (effects of enzyme overexpression or knockouts on the steady state production rate to screen possible models. These data are routinely generated during strain design. An ensemble of models is constructed that all reach the same steady state and are based on the same mechanistic framework at the elementary reaction level. The behavior of the models spans the kinetics allowable by thermodynamics. Then by using existing data from the literature for the overexpression of genes coding for transketolase (Tkt, transaldolase (Tal, and phosphoenolpyruvate synthase (Pps to screen the ensemble, we arrive at a set of models that properly describes the known enzyme overexpression phenotypes. This subset of models becomes more predictive as additional data are used to refine the models. The final ensemble of models demonstrates the characteristic of the cell that Tkt is the first rate controlling step, and correctly predicts that only after Tkt is overexpressed does an increase in Pps increase the production rate of aromatics. This work demonstrates that EM is able to capture the result of enzyme overexpression on aromatic producing bacteria by successfully utilizing routinely generated enzyme tuning data to guide model learning.

Cloning, expression and characterization of two S-ribosylhomocysteine lyases from Lactobacillus plantarum YM-4-3: Implication of conserved and divergent roles in quorum sensing.

Science.gov (United States)

Song, Xiao-Dong; Liu, Chen-Jian; Huang, Shi-Hao; Li, Xiao-Ran; Yang, En; Luo, Yi-Yong

2018-05-01

Quorum sensing (QS) is a means of cell-to-cell communication that regulates, via small signalling molecules, expression of a series of genes and controls multicellular behaviour in many bacterial species. The enzyme S-ribosylhomocysteine lyase (LuxS) transforms S-ribosylhomocysteine (SRH) into 4, 5-dihydroxy-2, 3-pentanedione (DPD), the precursor of the interspecies QS signalling molecule autoinducer-2 (AI-2). In this study, two LuxS-coding genes, luxS1 and luxS2, with 70% sequence identity were isolated from Lactobacillus plantarum YM-4-3, and overexpressed in Escherichia coli BL21 (DE3), and the protein products were purified successfully. After incubation of LuxS1 or LuxS2 with SRH, the reaction products were able to induce Vibrio harveyi BB170 bioluminescence, clearly demonstrating that both LuxS1 and LuxS2 synthesize AI-2 from SRH in vitro. Ellman's assay results revealed optimal temperatures for LuxS1 and LuxS2 of 45 and 37 °C, respectively, and their activities were stimulated or inhibited by several metal ions and chemical reagents. In addition, enzyme kinetics data showed that K m , V max and K cat value of LuxS1 for the substrate (SRH) were higher than that of LuxS2. These results suggest that LuxS1 and LuxS2 mediate QS in a temperature-dependent manner and may play conserved roles in AI-2 synthesis but exhibit different activities in response to external environmental stress. To our knowledge, this paper is the first report of two luxS genes present in one bacterial genome and the subsequent comparative elucidation of their functions in AI-2 production. Collectively, our study provides a solid basis for future research concerning the AI-2/LuxS QS system in L. plantarum YM-4-3. Copyright © 2018 Elsevier Inc. All rights reserved.

Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions

DEFF Research Database (Denmark)

Bonomo, Silvia; Jorgensen, Flemming Steen; Olsen, Lars

2017-01-01

Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17–C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have characte...... the concept that the selectivity of the steroidogenic CYPs is ruled by direct interactions with the enzyme, in contrast to the selectivity of drug-metabolizing CYPs, where the reactivity of the substrates dominates....... characterized the reaction path for both the hydroxylase and lyase reactions using density functional theory (DFT) calculations and the enzyme–substrate interactions by molecular dynamics (MD) simulations. Activation barriers for positions subject to hydroxylase reaction have values close to each other and span...

Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

Science.gov (United States)

Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

2014-11-07

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Arsenic Demethylation by a C·As Lyase in Cyanobacterium Nostoc sp. PCC 7120.

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Yan, Yu; Ye, Jun; Xue, Xi-Mei; Zhu, Yong-Guan

2015-12-15

Arsenic, a ubiquitous toxic substance, exists mainly as inorganic forms in the environment. It is perceived that organoarsenicals can be demethylated and degraded into inorganic arsenic by microorganisms. Few studies have focused on the mechanism of arsenic demethylation in bacteria. Here, we investigated arsenic demethylation in a typical freshwater cyanobacterium Nostoc sp. PCC 7120. This bacterium was able to demethylate monomethylarsenite [MAs(III)] rapidly to arsenite [As(III)] and also had the ability to demethylate monomethylarsenate [MAs(V)] to As(III). The NsarsI encoding a C·As lyase responsible for MAs(III) demethylation was cloned from Nostoc sp. PCC 7120 and heterologously expressed in an As-hypersensitive strain Escherichia coli AW3110 (ΔarsRBC). Expression of NsarsI was shown to confer MAs(III) resistance through arsenic demethylation. The purified NsArsI was further identified and functionally characterized in vitro. NsArsI existed mainly as the trimeric state, and the kinetic data were well-fit to the Hill equation with K0.5 = 7.55 ± 0.33 μM for MAs(III), Vmax = 0.79 ± 0.02 μM min(-1), and h = 2.7. Both of the NsArsI truncated derivatives lacking the C-terminal 10 residues (ArsI10) or 23 residues (ArsI23) had a reduced ability of MAs(III) demethylation. These results provide new insights for understanding the important role of cyanobacteria in arsenic biogeochemical cycling in the environment.

Hydrogen Peroxide Treatment and the Phenylpropanoid Pathway Precursors Feeding Improve Phenolics and Antioxidant Capacity of Quinoa Sprouts via an Induction of L-Tyrosine and L-Phenylalanine Ammonia-Lyases Activities

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Michał Świeca

2016-01-01

Full Text Available Hydrogen peroxide treatment and the phenylpropanoid pathway precursors feeding affected the antioxidant capacity of quinoa sprouts. Compared to the control, total phenolics content was significantly increased by treatment of control sprouts with 50 mM and 200 mM H2O2—an elevation of about 24% and 28%, respectively. The highest increase of flavonoids content was found for the sprouts treated with 200 mM H2O2 obtained from seeds fed with shikimic acid. All the studied modifications increased the antioxidant potential of sprouts (at least by 50% compared to control. The highest reducing power was found for the sprouts treated with 200 mM H2O2 obtained by phenylalanine feeding (5.03 mg TE/g DW and those obtained from the seeds fed with tyrosine (5.26 mg TE/g DW. The activities of L-tyrosine (TAL and L-phenylalanine (PAL ammonia-lyases were strongly affected by germination time as well as the applied modification of sprouting. On the 3rd day the highest PAL activity was determined for both untreated and induced with 50 mM H2O2 sprouts obtained by phenylalanine feeding. H2O2 induced TAL activity; the highest TAL activity was determined for 3-day-old sprouts induced with 200 mM H2O2 obtained from seeds fed with phenylalanine.

Metabolic engineering of Pseudomonas putida KT2440 for the production of para-hydroxy benzoic acid

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Shiqin Yu

2016-11-01

Full Text Available para-hydroxy benzoic acid (PHBA is the key component for preparing parabens, a common preservatives in food, drugs and personal care products, as well as high performance bioplastics such as liquid crystal polymers (LCP. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA or competing for the precursor chorismate (pheA and trpE were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose-4- phosphate and NAPH supply. The best strain achieved a maximum titre of 1.73 g L-1 and a carbon yield of 18.1 % (C-mol C-mol-1 in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase.

Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells

International Nuclear Information System (INIS)

Pei, Yanxi; Wu, Bo; Cao, Qiuhui; Wu, Lingyun; Yang, Guangdong

2011-01-01

Hydrogen sulfide (H 2 S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H 2 S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H 2 S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H 2 S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H 2 S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H 2 S-producing enzyme in prostate. CSE overexpression enhanced H 2 S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H 2 S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H 2 S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H 2 S-releasing diet or drug might be beneficial in the treatment of prostate cancer. Highlights: ► A large amount of H 2 S is released from sulforaphane. ► H 2 S mediates the anti-survival effect of sulforaphane on human prostate cancer cells. ► Cystathionine gamma-lyase is a major H 2 S

AlgM4: A New Salt-Activated Alginate Lyase of the PL7 Family with Endolytic Activity

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Guiyuan Huang

2018-04-01

Full Text Available Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7. In this study, the kinetic constants Vmax (maximum reaction rate and Km (Michaelis constant of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the β-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM, and poly-guluronic acid (polyG, resulting in the production of oligosaccharides with a degree of polymerization (DP of 2–9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

NARCIS (Netherlands)

Hussain, Z.; Wiedner, R.; Steiner, K.; Hajek, T.; Avi, M.; Hecher, B.; Sessitsch, A.; Schwab, H.

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity

Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

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Santiago, André S; Santos, Clelton A; Mendes, Juliano S; Toledo, Marcelo A S; Beloti, Lilian L; Souza, Alessandra A; Souza, Anete P

2015-09-01

The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/β fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrogen sulfide ameliorated L-NAME-induced hypertensive heart disease by the Akt/eNOS/NO pathway.

Science.gov (United States)

Jin, Sheng; Teng, Xu; Xiao, Lin; Xue, Hongmei; Guo, Qi; Duan, Xiaocui; Chen, Yuhong; Wu, Yuming

2017-12-01

Reductions in hydrogen sulfide (H 2 S) production have been implicated in the pathogenesis of hypertension; however, no studies have examined the functional role of hydrogen sulfide in hypertensive heart disease. We hypothesized that the endogenous production of hydrogen sulfide would be reduced and exogenous hydrogen sulfide would ameliorate cardiac dysfunction in N ω -nitro- L-arginine methyl ester ( L-NAME)-induced hypertensive rats. Therefore, this study investigated the cardioprotective effects of hydrogen sulfide on L-NAME-induced hypertensive heart disease and explored potential mechanisms. The rats were randomly divided into five groups: Control, Control + sodium hydrosulfide (NaHS), L-NAME, L-NAME + NaHS, and L-NAME + NaHS + glibenclamide (Gli) groups. Systolic blood pressure was monitored each week. In Langendorff-isolated rat heart, cardiac function represented by ±LV dP/dt max and left ventricular developing pressure was recorded after five weeks of treatment. Hematoxylin and Eosin and Masson's trichrome staining and myocardium ultrastructure under transmission electron microscopy were used to evaluate cardiac remodeling. The plasma nitric oxide and hydrogen sulfide concentrations, as well as nitric oxide synthases and cystathionine-γ-lyase activity in left ventricle tissue were determined. The protein expression of p-Akt, Akt, p-eNOS, and eNOS in left ventricle tissue was analyzed using Western blot. After five weeks of L-NAME treatment, there was a time-dependent hypertension, cardiac remodeling, and dysfunction accompanied by a decrease in eNOS phosphorylation, nitric oxide synthase activity, and nitric oxide concentration. Meanwhile, cystathionine-γ-lyase activity and hydrogen sulfide concentration were also decreased. NaHS treatment significantly increased plasma hydrogen sulfide concentration and subsequently promoted the Akt/eNOS/NO pathway which inhibited the development of hypertension and attenuated cardiac remodeling and

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

Science.gov (United States)

Bogani, Federica; Boehmer, Paul E.

2008-01-01

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5′-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3′ side of abasic sites and 5′-dRP residues that remain after cleavage by 5′-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency. PMID:18695225

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency.

Science.gov (United States)

Stuy, M; Chen, G-F; Masonek, J M; Scharschmidt, B F

2015-09-01

A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency☆

Science.gov (United States)

Stuy, M.; Chen, G.-F.; Masonek, J.M.; Scharschmidt, B.F.

2015-01-01

A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet. PMID:26937403

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Science.gov (United States)

Weske, Sarah; Vaidya, Mithila; Reese, Alina; von Wnuck Lipinski, Karin; Keul, Petra; Bayer, Julia K; Fischer, Jens W; Flögel, Ulrich; Nelsen, Jens; Epple, Matthias; Scatena, Marta; Schwedhelm, Edzard; Dörr, Marcus; Völzke, Henry; Moritz, Eileen; Hannemann, Anke; Rauch, Bernhard H; Gräler, Markus H; Heusch, Gerd; Levkau, Bodo

2018-05-01

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P 2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P 2 -deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

Science.gov (United States)

Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

2012-03-01

Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

Science.gov (United States)

Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

2008-04-01

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide

Directory of Open Access Journals (Sweden)

Haifeng Li

2018-05-01

Full Text Available An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL in yeast culture supernatant reached 226.4 μg/mL (915.5 U/mL. This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K+ ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T5015 of 57–58 °C and T5030 of 53–54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by P. pastoris attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using P. pastoris system.

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

Science.gov (United States)

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.

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Lee, Youngdeuk; Oh, Chulhong; De Zoysa, Mahanama; Kim, Hyowon; Wickramaarachchi, Wickramaarachchige Don Niroshana; Whang, Ilson; Kang, Do-Hyung; Lee, Jehee

2013-01-01

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency

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M. Stuy

2015-09-01

Full Text Available A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB. The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

Overexpressed Proteins in Hypervirulent Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 Compared to E. coli O157:H7 EDL933 Clade 3 Strain.

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Natalia Amigo

Full Text Available Escherichia coli O157:H7 is responsible for severe diarrhea and hemolytic uremic syndrome (HUS, and predominantly affects children under 5 years. The major virulence traits are Shiga toxins, necessary to develop HUS and the Type III Secretion System (T3SS through which bacteria translocate effector proteins directly into the host cell. By SNPs typing, E. coli O157:H7 was separated into nine different clades. Clade 8 and clade 6 strains were more frequently associated with severe disease and HUS. In this study, we aimed to identify differentially expressed proteins in two strains of E. coli O157:H7 (clade 8 and clade 6, obtained from cattle and compared them with the well characterized reference EDL933 strain (clade 3. Clade 8 and clade 6 strains show enhanced pathogenicity in a mouse model and virulence-related properties. Proteins were extracted and analyzed using the TMT-6plex labeling strategy associated with two dimensional liquid chromatography and mass spectrometry in tandem. We detected 2241 proteins in the cell extract and 1787 proteins in the culture supernatants. Attention was focused on the proteins related to virulence, overexpressed in clade 6 and 8 strains compared to EDL933 strain. The proteins relevant overexpressed in clade 8 strain were the curli protein CsgC, a transcriptional activator (PchE, phage proteins, Stx2, FlgM and FlgD, a dienelactone hydrolase, CheW and CheY, and the SPATE protease EspP. For clade 6 strain, a high overexpression of phage proteins was detected, mostly from Stx2 encoding phage, including Stx2, flagellin and the protease TagA, EDL933_p0016, dienelactone hydrolase, and Haemolysin A, amongst others with unknown function. Some of these proteins were analyzed by RT-qPCR to corroborate the proteomic data. Clade 6 and clade 8 strains showed enhanced transcription of 10 out of 12 genes compared to EDL933. These results may provide new insights in E. coli O157:H7 mechanisms of pathogenesis.

The effect of hydrogen peroxide and ultraviolet irradiation on non-sporing bacteria

International Nuclear Information System (INIS)

Bayliss, C.E.; Waites, W.M.

1980-01-01

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0%(w/v) for 75 and 180 min respectively. The same kill was produced by 30s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide. (author)

Protective Effects of Hydrogen Sulfide in the Ageing Kidney.

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Hou, Cui-Lan; Wang, Ming-Jie; Sun, Chen; Huang, Yong; Jin, Sheng; Mu, Xue-Pan; Chen, Ying; Zhu, Yi-Chun

2016-01-01

Aims . The study aimed to examine whether hydrogen sulfide (H 2 S) generation changed in the kidney of the ageing mouse and its relationship with impaired kidney function. Results . H 2 S levels in the plasma, urine, and kidney decreased significantly in ageing mice. The expression of two known H 2 S-producing enzymes in kidney, cystathionine γ -lyase (CSE) and cystathionine- β -synthase (CBS), decreased significantly during ageing. Chronic H 2 S donor (NaHS, 50  μ mol/kg/day, 10 weeks) treatment could alleviate oxidative stress levels and renal tubular interstitial collagen deposition. These protective effects may relate to transcription factor Nrf2 activation and antioxidant proteins such as HO-1, SIRT1, SOD1, and SOD2 expression upregulation in the ageing kidney after NaHS treatment. Furthermore, the expression of H 2 S-producing enzymes changed with exogenous H 2 S administration and contributed to elevated H 2 S levels in the ageing kidney. Conclusions . Endogenous hydrogen sulfide production in the ageing kidney is insufficient. Exogenous H 2 S can partially rescue ageing-related kidney dysfunction by reducing oxidative stress, decreasing collagen deposition, and enhancing Nrf2 nuclear translocation. Recovery of endogenous hydrogen sulfide production may also contribute to the beneficial effects of NaHS treatment.

Enhancement of L-cysteine production by disruption of yciW in Escherichia coli.

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Kawano, Yusuke; Ohtsu, Iwao; Takumi, Kazuhiro; Tamakoshi, Ai; Nonaka, Gen; Funahashi, Eri; Ihara, Masaki; Takagi, Hiroshi

2015-02-01

Using in silico analysis, the yciW gene of Escherichia coli was identified as a novel L-cysteine regulon that may be regulated by the transcriptional activator CysB for sulfur metabolic genes. We found that overexpression of yciW conferred tolerance to L-cysteine, but disruption of yciW increased L-cysteine production in E. coli. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

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Li Mingji

2012-02-01

Full Text Available Abstract Background For metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-tac-promoters (MCPtacs in tandem. Results Through a uniquely designed in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB production proved that it was efficient. Integration of the phaCAB genes with the 5CPtacs promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation. Conclusions The transcription strength of the MCPtacs promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtacs promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.

Engineering of Escherichia coli for the synthesis of N-hydroxycinnamoyl tryptamine and serotonin.

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Lee, Su Jin; Sim, Geun-Young; Lee, Youngshim; Kim, Bong-Gyu; Ahn, Joong-Hoon

2017-11-01

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC-tryptamines and HC-serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.

H2S: a universal defense against antibiotics in bacteria.

Science.gov (United States)

Shatalin, Konstantin; Shatalina, Elena; Mironov, Alexander; Nudler, Evgeny

2011-11-18

Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

International Nuclear Information System (INIS)

Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro

1989-01-01

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M r of its subunit was 77,000. The cells converted [ 14 C]-L-phenylalanine into [ 14 C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M r 77,137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology

Overexpression of functional human oxidosqualene cyclase in Escherichia coli

DEFF Research Database (Denmark)

Kürten, Charlotte; Uhlén, Mathias; Syrén, Per-Olof

2015-01-01

The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the te......The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation...... of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h...... of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells...

New lupane triterpenoids from Solidago canadensis that inhibit the lyase activity of DNA polymerase beta.

Science.gov (United States)

Chaturvedula, V S Prakash; Zhou, Bing-Nan; Gao, Zhijie; Thomas, Shannon J; Hecht, Sidney M; Kingston, David G I

2004-12-01

Bioassay-directed fractionation of a methyl ethyl ketone extract of Solidago canadensis L. (Asteraceae), using an assay to detect the lyase activity of DNA polymerase beta, resulted in the isolation of the four new lupane triterpenoids 1-4 and the seven known compounds lupeol, lupeyl acetate, ursolic acid, cycloartenol, cycloartenyl palmitate, alpha-amyrin acetate, and stigmasterol. The structures of the new compounds were established as 3beta-(3R-acetoxyhexadecanoyloxy)-lup-20(29)-ene (1), 3beta-(3-ketohexadecanoyloxy)-lup-20(29)-ene (2), 3beta-(3R-acetoxyhexadecanoyloxy)-29-nor-lupan-20-one (3), and 3beta-(3-hetohexadecanoyloxy)-29-nor-lupan-20-one (4), respectively, on the basis of extensive 1D and 2D NMR spectroscopic interpretation and chemical modification studies. All 11 compounds were inhibitory to the lyase activity of DNA polymerase beta.

Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

International Nuclear Information System (INIS)

Suzuki, Ryuichiro; Kim, Byung-Jun; Shibata, Tsuyoshi; Iwamoto, Yuki; Katayama, Takane; Ashida, Hisashi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya; Yamamoto, Kenji

2010-01-01

Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl 2 , 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon

International Nuclear Information System (INIS)

Torres, Rodrigo; Chim, Nicholas; Sankaran, Banumathi; Pujol, Céline; Bliska, James B.; Goulding, Celia W.

2011-01-01

Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding. Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestisrip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α) 8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein

Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial.

Science.gov (United States)

Longo, Nicola; Harding, Cary O; Burton, Barbara K; Grange, Dorothy K; Vockley, Jerry; Wasserstein, Melissa; Rice, Gregory M; Dorenbaum, Alejandro; Neuenburg, Jutta K; Musson, Donald G; Gu, Zhonghua; Sile, Saba

2014-07-05

Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. Phenylalanine ammonia lyase is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. We aimed to assess the safety, tolerability, pharmacokinetic characteristics, and efficacy of recombinant Anabaena variabilis phenylalanine ammonia lyase (produced in Escherichia coli) conjugated with polyethylene glycol (rAvPAL-PEG) in reducing phenylalanine concentrations in adult patients with phenylketonuria. In this open-label, phase 1, multicentre trial, single subcutaneous injections of rAvPAL-PEG were given in escalating doses (0·001, 0·003, 0·010, 0·030, and 0·100 mg/kg) to adults with phenylketonuria. Participants aged 18 years or older with blood phenylalanine concentrations of 600 μmol/L or higher were recruited from among patients attending metabolic disease clinics in the USA. The primary endpoints were safety and tolerability of rAvPAL-PEG. Secondary endpoints were the pharmacokinetic characteristics of the drug and its effect on concentrations of phenylalanine. Participants and investigators were not masked to assigned dose group. This study is registered with ClinicalTrials.gov, number NCT00925054. 25 participants were recruited from seven centres between May 6, 2008, and April 15, 2009, with five participants assigned to each escalating dose group. All participants were included in the safety population. The most frequently reported adverse events were injection-site reactions and dizziness, which were self-limited and without sequelae. Two participants had serious adverse reactions to intramuscular medroxyprogesterone acetate, a drug that contains polyethylene glycol as an excipient. Three of five participants given the highest dose of rAvPAL-PEG (0·100 mg/kg) developed a generalised skin rash

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

International Nuclear Information System (INIS)

Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

2014-01-01

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

Energy Technology Data Exchange (ETDEWEB)

Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

2014-01-24

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Pei, Yanxi [Department of Biology, Lakehead University, Thunder Bay (Canada); College of Life Science, Shanxi University, Taiyuan (China); Wu, Bo [Department of Biology, Lakehead University, Thunder Bay (Canada); Department of Pathophysiology, Harbin Medical University, Harbin (China); Cao, Qiuhui [Department of Biology, Lakehead University, Thunder Bay (Canada); Wu, Lingyun [Department of Pathophysiology, Harbin Medical University, Harbin (China); Department of Pharmacology, University of Saskatchewan, Saskatoon (Canada); Yang, Guangdong, E-mail: gyang@lakeheadu.ca [The School of Kinesiology, Lakehead University, Thunder Bay (Canada)

2011-12-15

Hydrogen sulfide (H{sub 2}S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H{sub 2}S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H{sub 2}S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H{sub 2}S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H{sub 2}S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H{sub 2}S-producing enzyme in prostate. CSE overexpression enhanced H{sub 2}S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H{sub 2}S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H{sub 2}S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H{sub 2}S-releasing diet or drug might be beneficial in the treatment of prostate cancer. Highlights: Black-Right-Pointing-Pointer A large amount of H{sub 2}S is released from sulforaphane. Black-Right-Pointing-Pointer H{sub 2}S mediates the anti-survival effect of

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

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Mathias J Gerl

Full Text Available Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1 HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

Quantitative nature of overexpression experiments

Science.gov (United States)

Moriya, Hisao

2015-01-01

Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation associated with the degree of overexpression. PMID:26543202

Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD)

International Nuclear Information System (INIS)

Pandey, Nishant Kumar; Pal, Ravi Kant; Kashyap, Maruthi; Bhavesh, Neel Sarovar

2012-01-01

The Escherichia coli PapD-like protein (EcpD), from uropathogenic Escherichia coli (UPEC), which is a periplasmic chaperon of Yad fimbriae was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.67 Å resolution and belonged to space group C222 1 . Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å 3 Da −1 , with 59% solvent content. Initial phases were determined by molecular replacement

Exposure of E. coli to DNA-methylating agents impairs biofilm formation and invasion of eukaryotic cells via down regulation of the N-acetylneuraminate lyase NanA

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Pamela eDi Pasquale

2016-02-01

Full Text Available DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analogue acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

One Year Experience of Pheburane® (Sodium Phenylbutyrate) Treatment in a Patient with Argininosuccinate Lyase Deficiency

OpenAIRE

Uçar, Sema Kalkan; Ozbaran, Burcu; Altinok, Yasemin Atik; Kose, Melis; Canda, Ebru; Kagnici, Mehtap; Coker, Mahmut

2015-01-01

Argininosuccinate lyase deficiency (ASLD) is a urea cycle disorder (UCD) treated with dietary adjustment and nitrogen scavenging agents. “Pheburane®” is a new tasteless and odour-free formulation of sodium phenylbutyrate, indicated in the treatment of UCD.

Induced resistance to hydrogen peroxide, UV and gamma radiation in bacillus species

International Nuclear Information System (INIS)

Bashandy, A.S.

2005-01-01

The catalase activity produced in four bacillus spp.(bacillus cereus, B. laterosporus, B. pumilus and B. subtilis (Escherichia coli was used for comparison) was measured and the sensitivity of these bacteria to hydrogen peroxide was tested. Bacillus spp. had higher resistance to hydrogen peroxide than E. coil. cultures of bacillus spp . When pretreated with sublethal level of hydrogen peroxide, became relatively resistant to the lethal effects of hydrogen than untreated control cultures. These pretreated cells were also resistant to lethality mediated by UV light and gamma radiation. The obtained results suggest that bacillus spp. Possess inducible defense mechanism (s) against the deleterious effects of oxidants and /or ionizing radiation

Strategies used for genetically modifying bacterial genome: ite-directed mutagenesis, gene inactivation, and gene over-expression*

Science.gov (United States)

Xu, Jian-zhong; Zhang, Wei-guo

2016-01-01

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

Isolation and characterization of an Antarctic Flavobacterium strain with agarase and alginate lyase activities

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Lavín Paris

2016-09-01

Full Text Available Several bacteria that are associated with macroalgae can use phycocolloids as a carbon source. Strain INACH002, isolated from decomposing Porphyra (Rhodophyta, in King George Island, Antarctica, was screened and characterized for the ability to produce agarase and alginate-lyase enzymatic activities. Our strain INACH002 was identified as a member of the genus Flavobacterium, closely related to Flavobacterium faecale, using 16S rRNA gene analysis. The INACH002 strain was characterized as psychrotrophic due to its optimal temperature (17ºC and maximum temperature (20°C of growth. Agarase and alginate-lyase displayed enzymatic activities within a range of 10°C to 50°C, with differences in the optimal temperature to hydrolyze agar (50°C, agarose (50°C and alginate (30°C during the first 30 min of activity. Strain Flavobacterium INACH002 is a promising Antarctic biotechnological resource; however, further research is required to illustrate the structural and functional bases of the enzymatic performance observed during the degradation of different substrates at different temperatures.

An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli.

Science.gov (United States)

Theodosiou, Eleni; Breisch, Marina; Julsing, Mattijs K; Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

2017-07-01

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na + /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc. �

Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

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Lin Yuheng

2012-04-01

Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

Change in the Content of Salicylic Acid and in the Activities of Phenylalanine Ammonia-Lyase and Catalase in Wheat Seedling Roots Under the Effect of Azospirillum Lectins

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Alen'kina S.A.

2012-05-01

Full Text Available We investigated the time course of changes in the endogenous content of salicylic acid, the ratio between the acid's free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of the lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum: A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3. Differences in plant response to the action of the lectins from these two strains were established. On the basis of the obtained data, a model was proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and to formation of induced resistance.

Expression of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B in Escherichia coli and Its Recovery from Inclusion Body

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Lalu Rudyat Telly Savalas

2017-12-01

Full Text Available The present study aims at expressing and partially purifying PtpB in active form. To achieve this, Mtb PtpB gene has been cloned into pET30a vector and overexpressed in Escherichia coli BL 21(DE3 under IPTG induction in the form of an inclusion body. Following resolubilization by urea and dialysis, the resulted PtpB has been shown to be active against para-Nitrophenyl phosphate.  It is concluded that the resulted PtpB has had been recovered from inclusion body to give the active form of the enzyme, and thus the success in overexpressing PtpB provides the required material to investigate the biochemical properties of the pathogen virulence factor further. 

Overexpression Analysis of emv2 gene coding for Late Embryogenesis Abundant Protein from Vigna radiata (Wilczek

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Rajesh S.

2008-10-01

Full Text Available Late embryogenesis abundant (LEA proteins are speculated to protect against water stress deficit in plants. An over expression system for mungbean late embryogenesis abundant protein, emv2 was constructed in a pET29a vector, designated pET-emv2 which is responsible for higher expression under the transcriptional/translational control of T7/lac promoter incorporated in the Escherichia coli BL21 (DE3.Induction protocol was optimized for pET recombinants harboring the target gene. Overexpressed EMV2 protein was purified to homogeneity and the protein profile monitored by SDS-PAGE.

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

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Francesca Fiory

Full Text Available The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15. In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Mode of action of pectin lyase A of Aspergillus niger on differently C6-substituted oligogalacturonides

NARCIS (Netherlands)

Alebeek, van G.J.W.M.; Christensen, T.M.I.E.; Schols, H.A.; Mikkelsen, J.D.; Voragen, A.G.J.

2002-01-01

A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C6-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of

Kinetic Resolution and Stereoselective Synthesis of 3-Substituted Aspartic Acids by Using Engineered Methylaspartate Ammonia Lyases

NARCIS (Netherlands)

Raj, Hans; Szymanski, Wiktor; Villiers, Jandré de; Puthan Veetil, Vinod; Quax, Wim J.; Shimamoto, Keiko; Janssen, Dick B.; Feringa, Ben L.; Poelarends, Gerrit J.

2013-01-01

Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described.

Exercise Increases Cystathionine-γ-lyase Expression and Decreases the Status of Oxidative Stress in Myocardium of Ovariectomized Rats.

Science.gov (United States)

Tang, Zhiping; Wang, Yujun; Zhu, Xiaoyan; Ni, Xin; Lu, Jianqiang

2016-01-01

Exercise could be a therapeutic approach for cardiovascular dysfunction induced by estrogen deficiency. Our previous study has shown that estrogen maintains cystathionine-γ-lyase (CSE) expression and inhibits oxidative stress in the myocardium of female rats. In the present study, we investigated whether exercise improves CSE expression and oxidative stress status and ameliorates isoproterenol (ISO)-induced cardiac damage in ovariectomized (OVX) rats. The results showed that treadmill training restored the ovariectomy-induced reduction of CSE and estrogen receptor (ER)α and decrease of total antioxidant capacity (T-AOC) and increase of malondialdehyde (MDA). The level of CSE was positively correlated to T-AOC and ERα while inversely correlated to MDA. OVX rats showed increases in the serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) and the percentage of TUNEL staining in myocardium upon ISO insult compared to sham rats. Exercise training significantly reduced the serum levels of LDH and CK and the percentage of TUNEL staining in myocardium upon ISO insult in OVX rats. In cultured cardiomyocytes, ISO treatment decreased cell viability and increased LDH release, while overexpression of CSE increased cell viability and decreased LDH release in the cells upon ISO insult. The results suggest that exercise training improves the oxidative stress status and ameliorates the cardiac damage induced by oxidative stress in OVX rats. The improvement of oxidative stress status by exercise might be at least partially due to upregulation of CSE/H2S signaling.

Significance of Aurora B overexpression in hepatocellular carcinoma. Aurora B Overexpression in HCC

International Nuclear Information System (INIS)

Lin, Zhong-Zhe; Jeng, Yung-Ming; Hu, Fu-Chang; Pan, Hung-Wei; Tsao, Hsin-Wei; Lai, Po-Lin; Lee, Po-Huang; Cheng, Ann-Lii; Hsu, Hey-Chi

2010-01-01

To investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC). The Aurora B and Aurora A mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the p53 and β-catenin genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of p53 and exon 3 of β-catenin. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines. Aurora B was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of Aurora B was associated with Aurora A overexpression (P = 0.0003) and p53 mutation (P = 0.002) and was inversely associated with β-catenin mutation (P = 0.002). Aurora B overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that Aurora B overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of p53 and β-catenin. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis. Aurora B overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death

Science.gov (United States)

Wang, Hao; Claudel, Patricia; Riemann, Michael; Hause, Bettina; Hugueney, Philippe; Nick, Peter

2018-01-01

Abstract Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity. PMID:29659985

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

OpenAIRE

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transform...

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

Science.gov (United States)

Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

2015-01-01

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Enantioselective Synthesis of Various Cyanohydrins Using Covalently Immobilized Preparations of Hydroxynitrile Lyase from Prunus dulcis.

Science.gov (United States)

Alagöz, Dilek; Tükel, S Seyhan; Yildirim, Deniz

2015-11-01

The carrier-based and carrier-free (cross-linked enzyme aggregate) covalent immobilizations of Prunus dulcis hydroxynitrile lyase were investigated. The immobilized preparations were tested for enantioselective carbon-carbon bond formation activity in the biphasic medium. Of the tested preparations, only cross-linked enzyme aggregate of P. dulcis hydroxynitrile lyase (PdHNL-CLEA) achieved the synthesis of (R)-mandelonitrile with 93% yield and 99% enantiopurity. PdHNL-CLEA was also used in the synthesis of various (R)-cyanohydrins from corresponding aldehydes/ketones and hydrocyanic acid. When 4-methoxybenzaldehyde, 4-methyl benzaldehyde, and 4-hydroxybenzaldehyde were used as substrates, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were obtained as 95-95, 85-79, and 2-25%, respectively, after 96 h at pH 4.0 and 5 °C. For acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, and 4-iodoacetophenone, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were 1-99, 20-84, 11-95, 5-99, and 3-24%, respectively at the same conditions. The results demonstrate PdHNL-CLEA can be effectively used in the synthesis of (R)-mandelonitrile.

PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937.

Science.gov (United States)

Nasser, William; Reverchon, Sylvie; Vedel, Regine; Boccara, Martine

2005-11-01

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.

Overexpression of OLE1 enhances stress tolerance and constitutively activates the MAPK HOG pathway in Saccharomyces cerevisiae.

Science.gov (United States)

Nasution, Olviyani; Lee, Young Mi; Kim, Eunjung; Lee, Yeji; Kim, Wankee; Choi, Wonja

2017-03-01

OLE1 of Saccharomyces cerevisiae encodes the sole and essential Δ-9 desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Upon ectopic overexpression of OLE1 in S. cerevisiae, significant increases in the membrane oleic acid content were observed. OLE1-overexpressing strains displayed enhanced tolerance to various stresses, better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide content. The OLE1-mediated enhanced stress tolerance was considerably diminished upon deletion of HOG1, which encodes the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway. Furthermore, OLE1 overexpression constitutively activated Hog1, which remained in the cytoplasm. Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets, including CTT1, HSP12, and STL1, and further, the cAMP and stress response elements present in the promoter. OLE1 overexpression neither caused nor relieved endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute to enhanced tolerance to various types of stress. Biotechnol. Bioeng. 2017;114: 620-631. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.

Science.gov (United States)

Lundgren, A; Leach, S; Tobias, J; Carlin, N; Gustafsson, B; Jertborn, M; Bourgeois, L; Walker, R; Holmgren, J; Svennerholm, A-M

2013-02-06

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. Copyright © 2013 Elsevier Ltd. All rights reserved.

MraZ from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

Energy Technology Data Exchange (ETDEWEB)

Adams, Melanie A.; Udell, Christian M.; Pal, Gour Pada; Jia, Zongchao, E-mail: jia@post.queensu.ca [Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6 (Canada)

2005-04-01

The crystallization and preliminary X-ray diffraction analysis of MraZ, formerly known as hypothetical protein YabB, from Escherichia coli K-12 is presented. The MraZ family of proteins, also referred to as the UPF0040 family, are highly conserved in bacteria and are thought to play a role in cell-wall biosynthesis and cell division. The murein region A (mra) gene cluster encodes MraZ proteins along with a number of other proteins involved in this complex process. To date, there has been no clear functional assignment provided for MraZ proteins and the structure of a homologue from Mycoplasma pneumoniae, MPN314, failed to suggest a molecular function. The b0081 gene from Escherichia coli that encodes the MraZ protein was cloned and the protein was overexpressed, purified and crystallized. This data is presented along with evidence that the E. coli homologue exists in a different oligomeric state to the MPN314 protein.

MraZ from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

International Nuclear Information System (INIS)

Adams, Melanie A.; Udell, Christian M.; Pal, Gour Pada; Jia, Zongchao

2005-01-01

The crystallization and preliminary X-ray diffraction analysis of MraZ, formerly known as hypothetical protein YabB, from Escherichia coli K-12 is presented. The MraZ family of proteins, also referred to as the UPF0040 family, are highly conserved in bacteria and are thought to play a role in cell-wall biosynthesis and cell division. The murein region A (mra) gene cluster encodes MraZ proteins along with a number of other proteins involved in this complex process. To date, there has been no clear functional assignment provided for MraZ proteins and the structure of a homologue from Mycoplasma pneumoniae, MPN314, failed to suggest a molecular function. The b0081 gene from Escherichia coli that encodes the MraZ protein was cloned and the protein was overexpressed, purified and crystallized. This data is presented along with evidence that the E. coli homologue exists in a different oligomeric state to the MPN314 protein

Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

Science.gov (United States)

Ouattara, Hadja D; Ouattara, Honoré G; Droux, Michel; Reverchon, Sylvie; Nasser, William; Niamke, Sébastien L

2017-09-01

Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pHfermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L.

Science.gov (United States)

Gomi, Kenji; Satoh, Masaru; Ozawa, Rika; Shinonaga, Yumi; Sanada, Sachiyo; Sasaki, Katsutomo; Matsumura, Masaya; Ohashi, Yuko; Kanno, Hiroo; Akimitsu, Kazuya; Takabayashi, Junji

2010-01-01

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice (Oryza sativa L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 (OsHPL2), an enzyme for producing C(6) volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C(6) volatile, (E)-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of (E)-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with (E)-2-hexenal induced resistance to bacterial blight. OsHPL2-overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived (E)-2-hexenal play some role in WBPH-induced resistance in rice.

Possible regulatory role of phenylalanine ammonia-lyase in the production of anthocyanins in asparagus (Asparagus officinalis L)

NARCIS (Netherlands)

Flores, F.B.; Oosterhaven, J.; Martinez-Madrid, M.C.; Romojaro, F.

2005-01-01

The regulatory role of phenylalanine ammonia-lyase (PAL) in the light-induced accumulation of anthocyanins in the epidermis of asparagus spears has been analysed. A correlation between the stimulation of PAL activity and the rise in total anthocyanin content has been observed. Light radiation

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the RNA polymerase domain of primase from Streptococcus mutans strain UA159

International Nuclear Information System (INIS)

Im, Dong-Won; Kim, Tae-O; Jung, Ha Yun; Oh, Ji Eun; Lee, Se Jin; Heo, Yong-Seok

2011-01-01

The RNA polymerase domain of primase from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 1.60 Å. Primase is the enzyme that synthesizes RNA primers on single-stranded DNA during normal DNA replication. In this study, the catalytic core domain of primase from Streptococcus mutans UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 1.60 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P4 1 or P4 3 , with unit-cell parameters a = b = 52.63, c = 110.31 Å. The asymmetric unit is likely to contain one molecule, with a corresponding V M of 1.77 Å 3 Da −1 and a solvent content of 30.7%

A Kinetic Platform to Determine the Fate of Hydrogen Peroxide in Escherichia coli.

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Kristin J Adolfsen

2015-11-01

Full Text Available Hydrogen peroxide (H2O2 is used by phagocytic cells of the innate immune response to kill engulfed bacteria. H2O2 diffuses freely into bacteria, where it can wreak havoc on sensitive biomolecules if it is not rapidly detoxified. Accordingly, bacteria have evolved numerous systems to defend themselves against H2O2, and the importance of these systems to pathogenesis has been substantiated by the many bacteria that require them to establish or sustain infections. The kinetic competition for H2O2 within bacteria is complex, which suggests that quantitative models will improve interpretation and prediction of network behavior. To date, such models have been of limited scope, and this inspired us to construct a quantitative, systems-level model of H2O2 detoxification in Escherichia coli that includes detoxification enzymes, H2O2-dependent transcriptional regulation, enzyme degradation, the Fenton reaction and damage caused by •OH, oxidation of biomolecules by H2O2, and repair processes. After using an iterative computational and experimental procedure to train the model, we leveraged it to predict how H2O2 detoxification would change in response to an environmental perturbation that pathogens encounter within host phagosomes, carbon source deprivation, which leads to translational inhibition and limited availability of NADH. We found that the model accurately predicted that NADH depletion would delay clearance at low H2O2 concentrations and that detoxification at higher concentrations would resemble that of carbon-replete conditions. These results suggest that protein synthesis during bolus H2O2 stress does not affect clearance dynamics and that access to catabolites only matters at low H2O2 concentrations. We anticipate that this model will serve as a computational tool for the quantitative exploration and dissection of oxidative stress in bacteria, and that the model and methods used to develop it will provide important templates for the

Cooperative functioning between phenylalanine ammonia lyase and isochorishmate synthase activities contributes to salicylic acid biosynthesis in soybean

Science.gov (United States)

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL), or the isochorishmate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We...

Genome-wide mapping of furfural tolerance genes in Escherichia coli.

Science.gov (United States)

Glebes, Tirzah Y; Sandoval, Nicholas R; Reeder, Philippa J; Schilling, Katherine D; Zhang, Min; Gill, Ryan T

2014-01-01

Advances in genomics have improved the ability to map complex genotype-to-phenotype relationships, like those required for engineering chemical tolerance. Here, we have applied the multiSCale Analysis of Library Enrichments (SCALEs; Lynch et al. (2007) Nat. Method.) approach to map, in parallel, the effect of increased dosage for >10(5) different fragments of the Escherichia coli genome onto furfural tolerance (furfural is a key toxin of lignocellulosic hydrolysate). Only 268 of >4,000 E. coli genes (∼ 6%) were enriched after growth selections in the presence of furfural. Several of the enriched genes were cloned and tested individually for their effect on furfural tolerance. Overexpression of thyA, lpcA, or groESL individually increased growth in the presence of furfural. Overexpression of lpcA, but not groESL or thyA, resulted in increased furfural reduction rate, a previously identified mechanism underlying furfural tolerance. We additionally show that plasmid-based expression of functional LpcA or GroESL is required to confer furfural tolerance. This study identifies new furfural tolerant genes, which can be applied in future strain design efforts focused on the production of fuels and chemicals from lignocellulosic hydrolysate.

Genome-wide mapping of furfural tolerance genes in Escherichia coli.

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Tirzah Y Glebes

Full Text Available Advances in genomics have improved the ability to map complex genotype-to-phenotype relationships, like those required for engineering chemical tolerance. Here, we have applied the multiSCale Analysis of Library Enrichments (SCALEs; Lynch et al. (2007 Nat. Method. approach to map, in parallel, the effect of increased dosage for >10(5 different fragments of the Escherichia coli genome onto furfural tolerance (furfural is a key toxin of lignocellulosic hydrolysate. Only 268 of >4,000 E. coli genes (∼ 6% were enriched after growth selections in the presence of furfural. Several of the enriched genes were cloned and tested individually for their effect on furfural tolerance. Overexpression of thyA, lpcA, or groESL individually increased growth in the presence of furfural. Overexpression of lpcA, but not groESL or thyA, resulted in increased furfural reduction rate, a previously identified mechanism underlying furfural tolerance. We additionally show that plasmid-based expression of functional LpcA or GroESL is required to confer furfural tolerance. This study identifies new furfural tolerant genes, which can be applied in future strain design efforts focused on the production of fuels and chemicals from lignocellulosic hydrolysate.

Immobilization of Escherichia coli containing ω‐transaminase activity in LentiKats®

DEFF Research Database (Denmark)

Cárdenas‐Fernández, Max; Lima Afonso Neto, Watson; López, Carmen

2012-01-01

Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized...... whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole...

Increased protein expression of LHCG receptor and 17a-hydroxylase/17,20-lyase in human polycystic ovaries

NARCIS (Netherlands)

Comim, F.V.; Teerds, K.J.; Hardy, K.; Franks, S.

2013-01-01

STUDY QUESTION Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? SUMMARY ANSWER LHCGR and 17a-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs).

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria.

Science.gov (United States)

Hu, Liyan; Pandey, Amit V; Balmer, Cécile; Eggimann, Sandra; Rüfenacht, Véronique; Nuoffer, Jean-Marc; Häberle, Johannes

2015-09-01

Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3% of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16% of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30% of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells.

Science.gov (United States)

McGoldrick, Trevor A; Lock, Edward A; Rodilla, Vicente; Hawksworth, Gabrielle M

2003-07-01

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached

IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

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Jand-Venes Rolim MEDEIROS

2013-04-01

Full Text Available Context Hydrogen sulphide (H2S has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. Objective To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE and cystathionine-β-synthetase (CBS isoforms in gastric mucosa of mice treated with saline or 50% ethanol. Methods Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g. After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. Results We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. Conclusion Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

Gene Overexpression: Uses, Mechanisms, and Interpretation

Science.gov (United States)

2012-01-01

The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease. PMID:22419077

Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Andersen, J T; Poulsen, Peter

1992-01-01

acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well....../min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than...

Towards an integrated system for bio-energy: hydrogen production by Escherichia coli and use of palladium-coated waste cells for electricity generation in a fuel cell.

Science.gov (United States)

Orozco, R L; Redwood, M D; Yong, P; Caldelari, I; Sargent, F; Macaskie, L E

2010-12-01

Escherichia coli strains MC4100 (parent) and a mutant strain derived from this (IC007) were evaluated for their ability to produce H(2) and organic acids (OAs) via fermentation. Following growth, each strain was coated with Pd(0) via bioreduction of Pd(II). Dried, sintered Pd-biomaterials ('Bio-Pd') were tested as anodes in a proton exchange membrane (PEM) fuel cell for their ability to generate electricity from H(2). Both strains produced hydrogen and OAs but 'palladised' cells of strain IC007 (Bio-Pd(IC007)) produced ~threefold more power as compared to Bio-Pd(MC4100) (56 and 18 mW respectively). The power output used, for comparison, commercial Pd(0) powder and Bio-Pd made from Desulfovibrio desulfuricans, was ~100 mW. The implications of these findings for an integrated energy generating process are discussed.

L-Cysteine Production in Escherichia coli Based on Rational Metabolic Engineering and Modular Strategy.

Science.gov (United States)

Liu, Han; Fang, Guochen; Wu, Hui; Li, Zhimin; Ye, Qin

2018-05-01

L-cysteine is an amino acid with important physiological functions and has a wide range of applications in medicine, food, animal feed, and cosmetics industry. In this study, the L-cysteine synthesis in Escherichia coliEscherichia coli is divided into four modules: the transport module, sulfur module, precursor module, and degradation module. The engineered strain LH03 (overexpression of the feedback-insensitive cysE and the exporter ydeD in JM109) accumulated 45.8 mg L -1 of L-cysteine in 48 hr with yield of 0.4% g/g glucose. Further modifications of strains and culture conditions which based on the rational metabolic engineering and modular strategy improved the L-cysteine biosynthesis significantly. The engineered strain LH06 (with additional overexpression of serA, serC, and serB and double mutant of tnaA and sdaA in LH03) produced 620.9 mg L -1 of L-cysteine with yield of 6.0% g/g glucose, which increased the production by 12 times and the yield by 14 times more than those of LH03 in the original condition. In fed-batch fermentation performed in a 5-L reactor, the concentration of L-cysteine achieved 5.1 g L -1 in 32 hr. This work demonstrates that the combination of rational metabolic engineering and module strategy is a promising approach for increasing the L-cysteine production in E. coli. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystallization and preliminary X-ray analysis of Escherichia coli RNase G

International Nuclear Information System (INIS)

Fang, Pengfei; Wang, Jing; Li, Xu; Guo, Min; Xing, Li; Cao, Xu; Zhu, Yi; Gao, Yan; Niu, Liwen; Teng, Maikun

2009-01-01

Full-length E. coli RNase G was overexpressed, purified and crystallized. Diffraction data were collected to a resolution of 3.4 Å. The homologous RNases RNase E and RNase G are widely distributed in bacteria and function in many important physiological processes, including mRNA degradation, rRNA maturation and so on. In this study, the crystallization and preliminary X-ray analysis of RNase G from Escherichia coli is described. Purified recombinant E. coli RNase G, which has 497 amino acids, was crystallized in the cubic space group F432, with unit-cell parameters a = b = c = 219.84 Å. X-ray diffraction data were collected to a resolution of 3.4 Å

Identification, expression, and characterization of a novel bacterial RGI Lyase enzyme for the production of bio-functional fibers

DEFF Research Database (Denmark)

da Silva, Ines Isabel Cardoso Rodrigues; Larsen, Dorte Møller; Meyer, Anne S.

2011-01-01

A gene encoding a putative rhamnogalacturonan I (RGI) Lyase (EC 4.2.2.-) from Bacillus licheniformis (DSM13) was selected after a homology search and phylogenetic analysis and optimized with respect to codon usage. The designed gene was transformed into Pichia pastoris and the enzyme was produced...

Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase*S⃞

Science.gov (United States)

Blot, Nicolas; Wu, Xian-Jun; Thomas, Jean-Claude; Zhang, Juan; Garczarek, Laurence; Böhm, Stephan; Tu, Jun-Ming; Zhou, Ming; Plöscher, Matthias; Eichacker, Lutz; Partensky, Frédéric; Scheer, Hugo; Zhao, Kai-Hong

2009-01-01

Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. PMID:19182270

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

Science.gov (United States)

Zechel, David L.; Jochimsen, Bjarne

2014-01-01

SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression

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Liangping Zha

2017-07-01

Full Text Available The content of active compounds differ in buds and flowers of Lonicera japonica (FLJ and L. japonica var. chinensis (rFLJ. Chlorogenic acid (CGAs were major active compounds of L. japonica and regarded as measurements for quality evaluation. However, little is known concerning the formation of active compounds at the molecular level. We quantified the major CGAs in FLJ and rFLJ, and found the concentrations of CGAs were higher in the buds of rFLJ than those of FLJ. Further analysis of CpG methylation of CGAs biosynthesis genes showed differences between FLJ and rFLJ in the 5′-UTR of phenylalanine ammonia-lyase 2 (PAL2. We identified 11 LjbZIP proteins and 24 rLjbZIP proteins with conserved basic leucine zipper domains, subcellular localization, and electrophoretic mobility shift assay showed that the transcription factor LjbZIP8 is a nuclear-localized protein that specifically binds to the G-box element of the LjPAL2 5′-UTR. Additionally, a transactivation assay and LjbZIP8 overexpression in transgenic tobacco indicated that LjbZIP8 could function as a repressor of transcription. Finally, treatment with 5-azacytidine decreased the transcription level of LjPAL2 and CGAs content in FLJ leaves. These results raise the possibility that DNA methylation might influence the recruitment of LjbZIP8, regulating PAL2 expression level and CGAs content in L. japonica.

The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

Science.gov (United States)

Mancini, Stefano; Imlay, James A

2015-05-01

Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology. © 2015 John Wiley & Sons Ltd.

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

International Nuclear Information System (INIS)

Bandhuvula, Padmavathi; Li Zaiguo; Bittman, Robert; Saba, Julie D.

2009-01-01

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K m of 35 μM for BODIPY-sphingosine 1-phosphate.

Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress.

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Ya-Dan Wen

Full Text Available BACKGROUND: Hydrogen sulfide (H₂S has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer's disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂ induced endothelial cells damage. METHODOLOGY AND PRINCIPAL FINDINGS: H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE, diphenyl-l-pyrenylphosphine (DPPP and malonaldehyde (MDA levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG, a selective inhibitor of cystathionine γ-lyase (CSE, abolished the protective effects of H₂S donors. INNOVATION: This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences. SIGNIFICANCE: H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.

The ketogenic diet is well tolerated and can be effective in patients with argininosuccinate lyase deficiency and refractory epilepsy

NARCIS (Netherlands)

Peuscher, Rosanne; Dijsselhof, Monique E.; Abeling, Nico G.; van Rijn, Margreet; van Spronsen, Francjan J.; Bosch, Annet M.

2012-01-01

Argininosuccinate lyase (ASL) deficiency (MIM 608310, McKusick 207900) is a rare disorder of the urea cycle, which leads to a deficiency of arginine and hyperammonemia. Epilepsy is a frequent complication of this disorder. A ketogenic diet (KD) can be a very effective therapy for refractory

The sequential action of a dipeptidase and a beta-lyase is required for the release of the human body odorant 3-methyl-3-sulfanylhexan-1-ol from a secreted Cys-Gly-(S) conjugate by Corynebacteria.

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Emter, Roger; Natsch, Andreas

2008-07-25

Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor.

Overexpression of Catalase in Vascular Smooth Muscle Cells Prevents the Formation of Abdominal Aortic Aneurysms

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Parastatidis, Ioannis; Weiss, Daiana; Joseph, Giji; Taylor, W Robert

2013-01-01

Objective Elevated levels of oxidative stress have been reported in abdominal aortic aneurysms (AAA), but which reactive oxygen species (ROS) promotes the development of AAA remains unclear. Here we investigate the effect of the hydrogen peroxide (H2O2) degrading enzyme catalase on the formation of AAA. Approach and Results AAA were induced with the application of calcium chloride (CaCl2) on mouse infrarenal aortas. The administration of PEG-catalase, but not saline, attenuated the loss of tunica media and protected against AAA formation (0.91±0.1 mm vs. 0.76±0.09 mm). Similarly, in a transgenic mouse model, catalase over-expression in the vascular smooth muscle cells (VSMC) preserved the thickness of tunica media and inhibited aortic dilatation by 50% (0.85±0.14 mm vs. 0.57±0.08 mm). Further studies showed that injury with CaCl2 decreased catalase expression and activity in the aortic wall. Pharmacologic administration or genetic over-expression of catalase restored catalase activity and subsequently decreased matrix metalloproteinase activity. In addition, a profound reduction in inflammatory markers and VSMC apoptosis was evident in aortas of catalase over-expressing mice. Interestingly, as opposed to infusion of PEG-catalase, chronic over-expression of catalase in VSMC did not alter the total aortic H2O2 levels. Conclusions The data suggest that a reduction in aortic wall catalase activity can predispose to AAA formation. Restoration of catalase activity in the vascular wall enhances aortic VSMC survival and prevents AAA formation primarily through modulation of matrix metalloproteinase activity. PMID:23950141

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase.

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Bamford, Vicki A; Armour, Maria; Mitchell, Sue A; Cartron, Michaël; Andrews, Simon C; Watson, Kimberly A

2008-09-01

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

NARCIS (Netherlands)

Rozeboom, Henriëtte J.; Yu, Shukun; Madrid, Susan; Kalk, Kor H.; Zhang, Ran; Dijkstra, Bauke W.

2013-01-01

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades

Epigenetic regulation of pro-inflammatory cytokine secretion by sphingosine 1-phosphate (S1P) in acute lung injury: Role of S1P lyase.

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Ebenezer, David L; Fu, Panfeng; Suryadevara, Vidyani; Zhao, Yutong; Natarajan, Viswanathan

2017-01-01

Cellular level of sphingosine-1-phosphate (S1P), the simplest bioactive sphingolipid, is tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and degradation mediated by S1P phosphatases, lipid phosphate phosphatases, and S1P lyase. The pleotropic actions of S1P are attributed to its unique inside-out (extracellular) signaling via G-protein-coupled S1P1-5 receptors, and intracellular receptor independent signaling. Additionally, S1P generated in the nucleus by nuclear SphK2 modulates HDAC1/2 activity, regulates histone acetylation, and transcription of pro-inflammatory genes. Here, we present data on the role of S1P lyase mediated S1P signaling in regulating LPS-induced inflammation in lung endothelium. Blocking S1P lyase expression or activity attenuated LPS-induced histone acetylation and secretion of pro-inflammatory cytokines. Degradation of S1P by S1P lyase generates Δ2-hexadecenal and ethanolamine phosphate and the long-chain fatty aldehyde produced in the cytoplasmic compartment of the endothelial cell seems to modulate histone acetylation pattern, which is different from the nuclear SphK2/S1P signaling and inhibition of HDAC1/2. These in vitro studies suggest that S1P derived long-chain fatty aldehyde may be an epigenetic regulator of pro-inflammatory genes in sepsis-induced lung inflammation. Trapping fatty aldehydes and other short chain aldehydes such as 4-hydroxynonenal derived from S1P degradation and lipid peroxidation, respectively by cell permeable agents such as phloretin or other aldehyde trapping agents may be useful in treating sepsis-induced lung inflammation via modulation of histone acetylation. . Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli

International Nuclear Information System (INIS)

Bakali H, M. Amin; Nordlund, Pär; Hallberg, B. Martin

2006-01-01

The overexpression, crystallization and preliminary diffraction analysis of E. coli YegS are reported. yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2 1 , with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 Å, β = 96.97°. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (V M ) of 2.5 Å 3 Da −1 and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 Å using synchrotron radiation

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice

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LI, XIHAI; LIANG, WENNA; YE, HONGZHI; WENG, XIAPING; LIU, FAYUAN; LIN, PINGDONG; LIU, XIANXIANG

2015-01-01

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice. PMID:26096903

Overexpressing the Sedum alfredii Cu/Zn Superoxide Dismutase Increased Resistance to Oxidative Stress in Transgenic Arabidopsis

Directory of Open Access Journals (Sweden)

Zhen Li

2017-06-01

Full Text Available Superoxide dismutase (SOD is a very important reactive oxygen species (ROS-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD, from Sedum alfredii, a cadmium (Cd/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2 and superoxide radicals (O2•-. The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress.

Cardiac-specific overexpression of catalase prevents diabetes-induced pathological changes by inhibiting NF-κB signaling activation in the heart.

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Cong, Weitao; Ruan, Dandan; Xuan, Yuanhu; Niu, Chao; Tao, Youli; Wang, Yang; Zhan, Kungao; Cai, Lu; Jin, Litai; Tan, Yi

2015-12-01

Catalase is an antioxidant enzyme that specifically catabolizes hydrogen peroxide (H2O2). Overexpression of catalase via a heart-specific promoter (CAT-TG) was reported to reduce diabetes-induced accumulation of reactive oxygen species (ROS) and further prevent diabetes-induced pathological abnormalities, including cardiac structural derangement and left ventricular abnormity in mice. However, the mechanism by which catalase overexpression protects heart function remains unclear. This study found that activation of a ROS-dependent NF-κB signaling pathway was downregulated in hearts of diabetic mice overexpressing catalase. In addition, catalase overexpression inhibited the significant increase in nitration levels of key enzymes involved in energy metabolism, including α-oxoglutarate dehydrogenase E1 component (α-KGD) and ATP synthase α and β subunits (ATP-α and ATP-β). To assess the effects of the NF-κB pathway activation on heart function, Bay11-7082, an inhibitor of the NF-κB signaling pathway, was injected into diabetic mice, protecting mice against the development of cardiac damage and increased nitrative modifications of key enzymes involved in energy metabolism. In conclusion, these findings demonstrated that catalase protects mouse hearts against diabetic cardiomyopathy, partially by suppressing NF-κB-dependent inflammatory responses and associated protein nitration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neuroprotective effects of lentivirus-mediated cystathionine-beta-synthase overexpression against 6-OHDA-induced parkinson's disease rats.

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Yin, Wei-Lan; Yin, Wei-Guo; Huang, Bai-Sheng; Wu, Li-Xiang

2017-09-14

Parkinson's disease (PD) is age-related neurodegenerative disorder by a progressive loss of dopaminergic(DA) neurons in the substantia nigra (SN) and striatum, which is at least partly associated with α-synuclein protein accumulation in these neurons. Hydrogen sulfide (H 2 S) plays an important role in the nervous system. Studies have shown that H 2 S has a protective effect on PD. However, as a kind of gas molecules, H 2 S is lively, volatile, and not conducive to scientific research and clinical application. Cystathionine-beta-synthase(CBS) is the main enzymes of synthesis of H 2 S in the brain. In order to examine the neuroprotective effects of CBS on PD, we detected the effects of CBS overexpression on 6-Hydroxydopamine (6-OHDA)-lesioned PD rats using lentivirus-mediated gene transfection techniques. In the injured SN of 6-OHDA-induced PD rats, the CBS expression and the endogenous H 2 S level markedly decreased, while administration of lentivirus-mediated CBS overexpression increased the CBS expression and the endogenous H 2 S production.CBS overexpression dramatically reversed apomorphine-induced rotation of the 6-OHDA model rats, decreased the number of TUNEL-positive neurons and the loss of the nigral DA neurons，specifically inhibited 6-OHDA-induced oxidase stress injury, and down-regulated the expression of α-synuclein(α-SYN) in the injured SN. NaHS (an H 2 S donor) had similar effects to CBS overexpression, while Amino-oxyacetate(AOAA, a CBS inhibitor) had opposite effects on PD rats. In summary, we demonstrated that CBS overexpression was able to provide neuroprotective on PD rats and improving the expression of CBS may be a potential therapeutic method for PD. Copyright © 2017. Published by Elsevier B.V.

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

Science.gov (United States)

Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Lin, Pingdong; Liu, Xianxiang

2015-09-01

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild‑type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression‑associated congenital dysplasia of the TMJ in mice.

Overexpression of artificially fused bifunctional enzyme 4CL1-CCR: a method for production of secreted 4-hydroxycinnamaldehydes in Escherichia coli.

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Liu, Shuxin; Qi, Qi; Chao, Nan; Hou, Jiayin; Rao, Guodong; Xie, Jin; Lu, Hai; Jiang, Xiangning; Gai, Ying

2015-08-12

4-Hydroxycinnamaldehydes are important intermediates in several secondary metabolism pathways, including those involved in the biosynthesis of phenolic acids, flavonoids, terpenoids and monolignols. They are also involved in the biosynthesis and degradation of lignins, which are important limiting factors during the processes of papermaking and biofuel production. Access to these aromatic polymers is necessary to explore the secondary biometabolic pathways they are involved in. Coniferaldehyde, sinapaldehyde, p-coumaraldehyde and caffealdehyde are members of the 4-hydroxycinnamaldehyde family. Although coniferaldehyde and sinapaldehyde can be purchased from commercial sources, p-coumaraldehyde and caffealdehyde are not commercially available. Therefore, there is increasing interest in producing 4-hydroxycinnamaldehydes. Here, we attempted to produce 4-hydroxycinnamaldehydes using engineered Escherichia coli. 4-Coumaric acid: coenzyme A ligase (4CL1) and cinnamoyl coenzyme A reductase (CCR) were fused by means of genetic engineering to generate an artificial bifunctional enzyme, 4CL1-CCR, which was overexpressed in cultured E. coli supplemented with phenylpropanoic acids. Three 4-hydroxycinnamaldehydes, p-coumaraldehyde, caffealdehyde and coniferaldehyde, were thereby biosynthesized and secreted into the culture medium. The products were extracted and purified from the culture medium, and identically characterized by the HPLC-PDA-ESI-MSn. The productivity of this new metabolic system were 49 mg/L for p-coumaraldehyde, 19 mg/L for caffealdehyde and 35 mg/L for coniferaldehyde. Extracellular hydroxycinnamoyl-coenzyme A thioesters were not detected, indicating that these thioesters could not pass freely through the cellular membrane. The fusion enzyme 4CL1-CCR can catalyze sequential multistep reactions, thereby avoiding the permeability problem of intermediates, which reveals its superiority over a mixture of individual native enzymes. Moreover, we have described a

C12 derivatives of the hydroperoxide lyase pathway are produced by product recycling through lipoxygenase-2 in Nicotiana attenuata leaves

NARCIS (Netherlands)

Kallenbach, M.; Gilardoni, P.A.; Allmann, S.; Baldwin, I.T.; Bonaventure, G.

2011-01-01

In response to diverse stresses, the hydroperoxide lyase (HPL) pathway produces C(6) aldehydes and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin). Since the original characterization of (10E )-traumatin and traumatic acid, little has been added to our knowledge of the metabolism and fluxes

NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia.

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Gonzalez Bosc, Laura V; Osmond, Jessica M; Giermakowska, Wieslawa K; Pace, Carolyn E; Riggs, Jennifer L; Jackson-Weaver, Olan; Kanagy, Nancy L

2017-04-01

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O 2 -5% CO 2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H 2 S)-induced vasodilation in rats. The enzyme that produces H 2 S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca 2+ concentration ([Ca 2+ ]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca 2+ ] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca 2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation. Copyright © 2017 the American Physiological Society.

C-MET overexpression and amplification in gliomas.

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Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

2015-01-01

We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

Production of itaconic acid from acetate by engineering acid-tolerant Escherichia coli W.

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Noh, Myung Hyun; Lim, Hyun Gyu; Woo, Sung Hwa; Song, Jinyi; Jung, Gyoo Yeol

2018-03-01

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli. © 2017 Wiley Periodicals, Inc.

¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

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Dany J V Beste

2011-07-01

Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

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Cheng Chung-Hsien

2009-07-01

Full Text Available Abstract Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D, and the other with five contiguous arginine residues (5R. Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac from N-acetyl-D-glucosamine (GlcNAc. Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulator AcrR from Escherichia coli

International Nuclear Information System (INIS)

Li, Ming; Qiu, Xi; Su, Chih-Chia; Long, Feng; Gu, Ruoyu; McDermott, Gerry; Yu, Edward W.

2006-01-01

The transcriptional regulator AcrR from Escherichia coli has been cloned, overexpressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.5 Å. This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å. The space group was determined to be P3 2 , with unit-cell parameters a = b = 46.61, c = 166.16 Å

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

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Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

2017-08-30

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

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Wu, Hong; Chen, Jinchun; Chen, Guo-Qiang

2016-12-01

E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

The effect of cadmium on phenylalanine ammonia lyase activity and lipid peroxidation in pepper (Capsicum annuum L. seedlings

Directory of Open Access Journals (Sweden)

Esra Koç

2015-04-01

Full Text Available In this study, the effect on differrent concentrations (20, 40, 80µM ve 100 µM CdCl2 of cadmium (CdCl2 on the activity of phenylalanine ammonia-lyase (PAL and lipid peroxidation amount in leaf and stem of Kahramanmaraş- Hot (Capsicum annum L. pepper seedlings were researched. Activity of phenylalanine ammonia-lyase (PAL, the first enzyme in the phenylpropanoid biosynthetic pathway, was increased at 2 and 4 days in KM-Hot plants exposed to CdCl2 stress. The highest PAL activity was detected in 20 μM CdCl2 application, on the four day after the application in the leaves of KM-Hot pepper. Moreover, it was observed that treatment of pepper with Cd led to an increased the rate of lipid peroxidation (which is indicated by increasing MDA content in the leaf and stem tissues. The highest MDA content was detected in 80 μM CdCl2 application, on the four day after the application in the leaf tissues. These results suggest that the activation of PAL may be associated with increased production of MDA

Mini-review: recent developments in hydroxynitrile lyases for industrial biotechnology.

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Lanfranchi, Elisa; Steiner, Kerstin; Glieder, Anton; Hajnal, Ivan; Sheldon, Roger A; van Pelt, Sander; Winkler, Margit

2013-12-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH.

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Tan, Hao; Wu, Xiang; Xie, Liyuan; Huang, Zhongqian; Gan, Bingcheng; Peng, Weihong

2015-06-01

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 μmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37 °C. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

MUREIN-METABOLIZING ENZYMES FROM ESCHERICHIA-COLI - SEQUENCE-ANALYSIS AND CONTROLLED OVEREXPRESSION OF THE SLT GENE, WHICH ENCODES THE SOLUBLE LYTIC TRANSGLYCOSYLASE

NARCIS (Netherlands)

ENGEL, H; KAZEMIER, B; KECK, W

The complete nucleotide sequence of the slt gene encoding the soluble lytic transglycosylase (Slt; EC 3.2.1.-) from Escherichia coli has been determined. The largest open reading frame identified on a 2.5-kb PvuII-SalI fragment indicates that the enzyme is translated as a preprotein of either 654 or

Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

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Patel, Arti T; Akhani, Rekha C; Patel, Manisha J; Dedania, Samir R; Patel, Darshan H

2017-06-01

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 μM and 2 M, respectively. The catalytic efficiency (k cat /K m ) for L-aspartic acid (14.18 s -1  mM -1 ) was higher than that for L-phenylalanine (4.65 s -1  mM -1 ). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 μM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

[Sudden death of a patient with 3-hydroxy-3-methylglutaryl coenzyme A lyase deficiency].

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Vilaseca Busca, M A; Ribes Rubio, A; Briones Godino, P; Cusi Sánchez, V; Baraíbar Castelló, R; Gairi Taull, J M

1990-02-01

A new case of neonatal 3-hydroxy-3-methylglutaric aciduria is described. 3-hydroxy-3-methylglutaryl CoA lyase activities in leukocytes demonstrated the patient's homozygosity and the heterozygous character of the parents and two other members of the family. Dietetic management with low fat high carbohydrate diet together with protein restriction and carnitine resulted in a good control of the metabolic acidosis, the hypoglycemia, and the physical and neurological development. Nevertheless, sudden death occurred at the age thirteen months without any previous apparent trouble and the necropsia showed neither signs of infection nor hepatic or cardiac derangement.

Kinetic resolution and stereoselective synthesis of 3-substituted aspartic acids by using engineered methylaspartate ammonia lyases.

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Raj, Hans; Szymanski, Wiktor; de Villiers, Jandré; Puthan Veetil, Vinod; Quax, Wim J; Shimamoto, Keiko; Janssen, Dick B; Feringa, Ben L; Poelarends, Gerrit J

2013-08-19

Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elimination of hydrogen sulphide and {beta} substitution in cystein, catalyzed by the cysteine-lyase of hens yolk-sac and yolk (1961); Desulfhydration et {beta} substitution de la cysteine catalysees par la cysteinelyase du sac vitellin et du jaune de l'oeuf de poule (1961)

Energy Technology Data Exchange (ETDEWEB)

Chapeville, F; Fromageot, P [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1961-07-01

The yolk of incubated hen's eggs contains a pyridoxal phosphate activated enzyme, free of iron, copper, magnesium and calcium. This enzyme activates the {beta}-carbon atom of cysteine. Its reactivity is demonstrated by the ease with which this {beta}-carbon fixes various sulfur containing substances in which the sulfur has reducing properties: inorganic sulfide, sulfide or cysteine itself. In the absence of substances able to react with the {beta}-carbon atom, the active complex, consisting of the enzyme and the aminated tri-carbon chain, is hydrolysed to pyruvic acid and ammonia. The liberation of hydrogen sulfide thus appears to be the consequence either of the substitution of the {beta}-carbon atom of cysteine or of the decomposition of the complex which this aminoacid forms with the enzyme studied. The latter seems therefore to possess an activity which differs from the activity of the desulfhydrases as yet known. We suggest to call this enzyme cystein-lyase. (authors) [French] Le sac vitellin et le jaune d'oeufs embryonnes de poule renferment une enzyme activee par le phosphate de pyridoxal, qui ne contient pas de fer, de magnesium, de cuivre ce de calcium et qui confere une reactivite particuliere au carbone {beta} de la cysteine. Cette reactivite se manifeste par l'aptitude que possede le carbone {beta} a fixer diverses molecules soufrees dont le soufre est reducteur, telles que le sulfure, le sulfite ou la cysteine elle-meme. En l'absence de reactifs capables de reagir avec le carbone {beta}, le complexe actif enzyme-chaine tricarbonee et aminee s'hydrolyse en acide pyruvique et en ammoniaque. La liberation d'hydrogene sulfure apparait ainsi comme une consequence soit de la substitution du carbone {beta} de la cysteine, soit de la decomposition du complexe qu'elle forme avec l'enzyme etudiee. Cette derniere semble donc posseder une activite distincte de celle des desulfhydrases connues jusqu'a present. Nous proposons de l'appeler cysteinelyase. (auteurs)

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

Science.gov (United States)

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Cadmium induced radioadaptive response via an ATM-independent H2S/cystathionine γ-lyase modulation

International Nuclear Information System (INIS)

Pan Yan; Yuan Dexiao; Zhang Jianghong; Shao Chunlin

2011-01-01

The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. Here we explored cadmium induced radioadaptive response (RAR) and investigated the role of hydrogen sulfide (H 2 S) and ATM kinase in this response. Our data showed that the cadmium ions with a sub-lethal concentration could induce RAR in Chang liver cells towards subsequent γ-irradiation and this response could be abrogated by DL-propargylglycine (PPG), the endogenous H 2 S synthetase inhibitor of cystathionine γ-lyase (CSE), but not by aminooxyacetic acid (AOAA), the inhibitor of cystathionine β-synthase (CBS). Moreover, the pretreatment of cells with NaHS also stimulated cellular adaptive response to radiation. Both cadmium treatment and irradiation up-regulated the expression of CSE protein in a time-dependent manner but had no influence on the expression of CBS protein. In the primed cells, the time course of CBS expression showed no significant difference with the cells treated with 2Gy irradiation alone, however, the CSE expression was easier to reach the maximum level, indicating a more efficient H 2 S production by CSE. Moreover, the cadmium-induced RAR was totally suppressed by KU-55933, a specific ATM inhibitor that did not change the CSE expression after radiation. However, exogenous H 2 S decreased the phosphorylation level of radiation-induced ATM. In conclusion, the present results demonstrate firstly that H 2 S is involved in the cadmium induced cross-adaptive response to challenging radiation. CSE, rather than CBS, may mainly responsible for the H 2 S production during this RAR which may also be mediated by ATM pathway. However, the activation of CSE is independent of ATM but could negatively regulate the phosphorylation of ATM.

Requirement of argininosuccinate lyase for systemic nitric oxide production.

Science.gov (United States)

Erez, Ayelet; Nagamani, Sandesh C S; Shchelochkov, Oleg A; Premkumar, Muralidhar H; Campeau, Philippe M; Chen, Yuqing; Garg, Harsha K; Li, Li; Mian, Asad; Bertin, Terry K; Black, Jennifer O; Zeng, Heng; Tang, Yaoping; Reddy, Anilkumar K; Summar, Marshall; O'Brien, William E; Harrison, David G; Mitch, William E; Marini, Juan C; Aschner, Judy L; Bryan, Nathan S; Lee, Brendan

2011-11-13

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

Science.gov (United States)

Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

2013-05-01

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

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Minako Sumiyoshi

Full Text Available Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5-linked l-Ara (LM6 and β-(1,4-linked d-Xyl (LM10 and LM11 residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.

Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii

International Nuclear Information System (INIS)

Chocat, P.; Esaki, N.; Tanizawa, K.; Nakamura, K.; Tanaka, H.; Soda, K.

1985-01-01

The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate. L-Cysteine is a competitive inhibitor of the enzyme. The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH 3 , pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme. However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar

Estimation of hydrogen production in genetically modified E. coli fermentations using an artificial neural network

Energy Technology Data Exchange (ETDEWEB)

Rosales-Colunga, Luis Manuel; De Leon Rodriguez, Antonio [Division de Biologia Molecular, Instituto Potosino de Investigacion Cientifica y Tecnologica, Camino a la Presa San Jose 2055, Col. Lomas 4a secc, San Luis Potosi, SLP 78216 (Mexico); Garcia, Raul Gonzalez [Centro de Investigacion y Estudios de Posgrado, Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi, Av. Dr. Manuel Nava 6, San Luis Potosi, SLP 78210 (Mexico)

2010-12-15

Biological hydrogen production is an active research area due to the importance of this gas as an energy carrier and the advantages of using biological systems to produce it. A cheap and practical on-line hydrogen determination is desired in those processes. In this study, an artificial neural network (ANN) was developed to estimate the hydrogen production in fermentative processes. A back propagation neural network (BPNN) of one hidden layer with 12 nodes was selected. The BPNN training was done using the conjugated gradient algorithm and on-line measurements of dissolved CO{sub 2}, pH and oxidation-reduction potential during the fermentations of cheese whey by Escherichia coli {delta}hycA {delta}lacI (WDHL) strain with or without pH control. The correlation coefficient between the hydrogen production determined by gas chromatography and the hydrogen production estimated by the BPNN was 0.955. Results showed that the BPNN successfully estimated the hydrogen production using only on-line parameters in genetically modified E. coli fermentations either with or without pH control. This approach could be used for other hydrogen production systems. (author)

Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

Science.gov (United States)

Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

2016-03-01

Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

Carbon and hydrogen isotopic effects of stomatal density in Arabidopsis thaliana

Science.gov (United States)

Lee, Hyejung; Feakins, Sarah J.; Sternberg, Leonel da S. L.

2016-04-01

Stomata are key gateways mediating carbon uptake and water loss from plants. Varied stomatal densities in fossil leaves raise the possibility that isotope effects associated with the openness of exchange may have mediated plant wax biomarker isotopic proxies for paleovegetation and paleoclimate in the geological record. Here we use Arabidopsis thaliana, a widely used model organism, to provide the first controlled tests of stomatal density on carbon and hydrogen isotopic compositions of cuticular waxes. Laboratory grown wildtype and mutants with suppressed and overexpressed stomatal densities allow us to directly test the isotope effects of stomatal densities independent of most other environmental or biological variables. Hydrogen isotope (D/H) measurements of both plant waters and plant wax n-alkanes allow us to directly constrain the isotopic effects of leaf water isotopic enrichment via transpiration and biosynthetic fractionations, which together determine the net fractionation between irrigation water and n-alkane hydrogen isotopic composition. We also measure carbon isotopic fractionations of n-alkanes and bulk leaf tissue associated with different stomatal densities. We find offsets of +15‰ for δD and -3‰ for δ13C for the overexpressed mutant compared to the suppressed mutant. Since the range of stomatal densities expressed is comparable to that found in extant plants and the Cenozoic fossil record, the results allow us to consider the magnitude of isotope effects that may be incurred by these plant adaptive responses. This study highlights the potential of genetic mutants to isolate individual isotope effects and add to our fundamental understanding of how genetics and physiology influence plant biochemicals including plant wax biomarkers.

Overexpression of a maize plasma membrane intrinsic protein ZmPIP1;1 confers drought and salt tolerance in Arabidopsis.

Science.gov (United States)

Zhou, Lian; Zhou, Jing; Xiong, Yuhan; Liu, Chaoxian; Wang, Jiuguang; Wang, Guoqiang; Cai, Yilin

2018-01-01

Drought and salt stress are major abiotic stress that inhibit plants growth and development, here we report a plasma membrane intrinsic protein ZmPIP1;1 from maize and identified its function in drought and salt tolerance in Arabidopsis. ZmPIP1;1 was localized to the plasma membrane and endoplasmic reticulum in maize protoplasts. Treatment with PEG or NaCl resulted in induced expression of ZmPIP1;1 in root and leaves. Constitutive overexpression of ZmPIP1;1 in transgenic Arabidopsis plants resulted in enhanced drought and salt stress tolerance compared to wild type. A number of stress responsive genes involved in cellular osmoprotection in ZmPIP1;1 overexpression plants were up-regulated under drought or salt condition. ZmPIP1;1 overexpression plants showed higher activities of reactive oxygen species (ROS) scavenging enzymes such as catalase and superoxide dismutase, lower contents of stress-induced ROS such as superoxide, hydrogen peroxide and malondialdehyde, and higher levels of proline under drought and salt stress than did wild type. ZmPIP1;1 may play a role in drought and salt stress tolerance by inducing of stress responsive genes and increasing of ROS scavenging enzymes activities, and could provide a valuable gene for further plant breeding.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

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Torres Leticia L

2012-08-01

Full Text Available Abstract Background Penicillin acylases (PACs are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1. The optimum pH was aprox. 4 and the optimum

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

Science.gov (United States)

Torres, Leticia L; Ferreras, Eloy R; Cantero, Angel; Hidalgo, Aurelio; Berenguer, José

2012-08-09

Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of

Sexual Dimorphism in the Selenocysteine Lyase Knockout Mouse.

Science.gov (United States)

Ogawa-Wong, Ashley N; Hashimoto, Ann C; Ha, Herena; Pitts, Matthew W; Seale, Lucia A; Berry, Marla J

2018-01-31

Selenium (Se) is an essential micronutrient known for its antioxidant properties and health benefits, attributed to its presence in selenoproteins as the amino acid, selenocysteine. Selenocysteine lyase (Scly) catalyzes hydrolysis of selenocysteine to selenide and alanine, facilitating re-utilization of Se for de novo selenoprotein synthesis. Previously, it was reported that male Scly -/- mice develop increased body weight and body fat composition, and altered lipid and carbohydrate metabolism, compared to wild type mice. Strikingly, females appeared to present with a less severe phenotype, suggesting the relationship between Scly and energy metabolism may be regulated in a sex-specific manner. Here, we report that while body weight and body fat gain occur in both male and female Scly -/- mice, strikingly, males are susceptible to developing glucose intolerance, whereas female Scly -/- mice are protected. Because Se is critical for male reproduction, we hypothesized that castration would attenuate the metabolic dysfunction observed in male Scly -/- mice by eliminating sequestration of Se in testes. We report that fasting serum insulin levels were significantly reduced in castrated males compared to controls, but islet area was unchanged between groups. Finally, both male and female Scly -/- mice exhibit reduced hypothalamic expression of selenoproteins S, M, and glutathione peroxidase 1.

Sexual Dimorphism in the Selenocysteine Lyase Knockout Mouse

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Ashley N. Ogawa-Wong

2018-01-01

Full Text Available Selenium (Se is an essential micronutrient known for its antioxidant properties and health benefits, attributed to its presence in selenoproteins as the amino acid, selenocysteine. Selenocysteine lyase (Scly catalyzes hydrolysis of selenocysteine to selenide and alanine, facilitating re-utilization of Se for de novo selenoprotein synthesis. Previously, it was reported that male Scly−/− mice develop increased body weight and body fat composition, and altered lipid and carbohydrate metabolism, compared to wild type mice. Strikingly, females appeared to present with a less severe phenotype, suggesting the relationship between Scly and energy metabolism may be regulated in a sex-specific manner. Here, we report that while body weight and body fat gain occur in both male and female Scly−/− mice, strikingly, males are susceptible to developing glucose intolerance, whereas female Scly−/− mice are protected. Because Se is critical for male reproduction, we hypothesized that castration would attenuate the metabolic dysfunction observed in male Scly−/− mice by eliminating sequestration of Se in testes. We report that fasting serum insulin levels were significantly reduced in castrated males compared to controls, but islet area was unchanged between groups. Finally, both male and female Scly−/− mice exhibit reduced hypothalamic expression of selenoproteins S, M, and glutathione peroxidase 1.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

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Chunxiang Yao

Full Text Available Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2, react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat, an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

Molecular cloning and cold shock induced overexpression of the DNA encoding phor sensor domain from Mycobacterium tuberculosis as a target molecule for novel anti-tubercular drugs

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Langi, Gladys Emmanuella Putri; Moeis, Maelita R.; Ihsanawati, Giri-Rachman, Ernawati Arifin

2014-03-01

Mycobacterium tuberculosis (Mtb), the sole cause of Tuberculosis (TB), is still a major global problem. The discovery of new anti-tubercular drugs is needed to face the increasing TB cases, especially to prevent the increase of cases with resistant Mtb. A potential novel drug target is the Mtb PhoR sensor domain protein which is the histidine kinase extracellular domain for receiving environmental signals. This protein is the initial part of the two-component system PhoR-PhoP regulating 114 genes related to the virulence of Mtb. In this study, the gene encoding PhoR sensor domain (SensPhoR) was subcloned from pGEM-T SensPhoR from the previous study (Suwanto, 2012) to pColdII. The construct pColdII SensPhoR was confirmed through restriction analysis and sequencing. Using the construct, SensPhoR was overexpressed at 15°C using Escherichia coli BL21 (DE3). Low temperature was chosen because according to the solubility prediction program of recombinant proteins from The University of Oklahama, the PhoR sensor domain has a chance of 79.8% to be expressed as insoluble proteins in Escherichia coli's (E. coli) cytoplasm. This prediction is also supported by other similar programs: PROSO and PROSO II. The SDS PAGE result indicated that the PhoR sensor domain recombinant protein was overexpressed. For future studies, this protein will be purified and used for structure analysis which can be used to find potential drugs through rational drug design.

Role of Hydrogen Sulfide in the Pathology of Inflammation

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Madhav Bhatia

2012-01-01

Full Text Available Hydrogen sulfide (H2S is a well-known toxic gas that is synthesized in the human body from the amino acids cystathionine, homocysteine, and cysteine by the action of at least two distinct enzymes: cystathionine-γ-lyase and cystathionine-β-synthase. In the past few years, H2S has emerged as a novel and increasingly important biological mediator. Imbalances in H2S have also been shown to be associated with various disease conditions. However, defining the precise pathophysiology of H2S is proving to be a complex challenge. Recent research in our laboratory has shown H2S as a novel mediator of inflammation and work in several groups worldwide is currently focused on determining the role of H2S in inflammation. H2S has been implicated in different inflammatory conditions, such as acute pancreatitis, sepsis, joint inflammation, and chronic obstructive pulmonary disease (COPD. Active research on the role of H2S in inflammation will unravel the pathophysiology of its actions in inflammatory conditions and may help develop novel therapeutic approaches for several, as yet incurable, disease conditions.

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium-Copy Number Plasmids in Escherichia coli.

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Ali, Syed A; Chew, Yik Wei

2015-01-01

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.

Engineering Saccharomyces cerevisiae To Release 3-Mercaptohexan-1-ol during Fermentation through Overexpression of an S. cerevisiae Gene, STR3, for Improvement of Wine Aroma▿

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Holt, Sylvester; Cordente, Antonio G.; Williams, Simon J.; Capone, Dimitra L.; Jitjaroen, Wanphen; Menz, Ian R.; Curtin, Chris; Anderson, Peter A.

2011-01-01

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5′-phosphate-dependent cystathionine β-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor. PMID:21478306

Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395

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Biswas, Maitree; Khamrui, Susmita; Sen, Udayaditya; Dasgupta, Jhimli

2011-01-01

The chemotaxis response regulator CheY4 from V. cholerae has been cloned, overexpressed, purified and crystallized in monoclinic and hexagonal space groups; the crystals diffracted to 1.67 and 1.9 Å resolution, respectively. Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1–CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni–NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, β = 96.5°, whereas crystals grown in space group P3 2 21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°

DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds

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Potapova, Olga; Chan, Chikio; DeLucia, Angela M.; Helquist, Sandra A.; Kool, Eric T.; Grindley, Nigel D. F.; Joyce, Catherine M.

2008-01-01

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair in order to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA Polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen bonding ability in the nascent pair, the efficiency (kpol/Kd) of the polymerase reaction is decreased by 30-fold, affecting ground state (Kd) and transition state (kpol) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. By contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions, and more dependent on hydrogen bonding between base-paired partners. PMID:16411765

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

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Yan, Wupeng; Stone, Everett; Zhang, Yan Jessie

2017-02-01

Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

Production of ethanol from thin stillage by metabolically engineered Escherichia coli.

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Gonzalez, Ramon; Campbell, Paul; Wong, Matthew

2010-03-01

Thin stillage is a by-product generated in large amounts during the production of ethanol that is rich in carbon sources like glycerol, glucose and maltose. Unfortunately, the fermentation of thin stillage results in a mixture of organic acids and ethanol and minimum utilization of glycerol, the latter a compound that can represent up to 80% of the available substrates in this stream. We report here the efficient production of ethanol from thin stillage by a metabolically engineered strain of Escherichia coli. Simultaneous utilization of glycerol and sugars was achieved by overexpressing either the fermentative or the respiratory glycerol-utilization pathway. However, amplification of the fermentative pathway (encoded by gldA and dhaKLM) led to more efficient consumption of glycerol and promoted the synthesis of reduced products, including ethanol. A previously constructed strain, EH05, containing mutations that prevented the accumulation of competing by-products (i.e. lactate, acetate, and succinate) and overexpressing the fermentative pathway for glycerol utilization [i.e. strain EH05 (pZSKLMgldA)], efficiently converted thin stillage supplemented with only mineral salts to ethanol at yields close to 85% of the theoretical maximum. Ethanol accounted for about 90% (w/w) of the product mixture. These results, along with the comparable performance of strain EH05 (pZSKLMgldA) in 0.5 and 5 l fermenters, indicate a great potential for the adoption of this process by the biofuels industry.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

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Steven Zhao

2016-10-01

Full Text Available Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY, cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

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Zhao, Steven; Torres, AnnMarie; Henry, Ryan A; Trefely, Sophie; Wallace, Martina; Lee, Joyce V; Carrer, Alessandro; Sengupta, Arjun; Campbell, Sydney L; Kuo, Yin-Ming; Frey, Alexander J; Meurs, Noah; Viola, John M; Blair, Ian A; Weljie, Aalim M; Metallo, Christian M; Snyder, Nathaniel W; Andrews, Andrew J; Wellen, Kathryn E

2016-10-18

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

International Nuclear Information System (INIS)

Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

2005-01-01

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli.

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Li, Xin; Imlay, James A

2018-03-14

Escherichia coli is a model organism that has been exploited to reveal key details of hydrogen peroxide stress: the biomolecules that H 2 O 2 most rapidly damages and the defensive tactics that organisms use to fend it off. Much less clear is the amount of exogenous H 2 O 2 that is sufficient to injure the bacterium and/or to trigger its stress response. To fill this gap, we need to study the behavior of cells when they are exposed to defined amounts of H 2 O 2 on an hours-long time scale. Such experiments are difficult because bacteria rapidly consume H 2 O 2 that is added to test cultures. Further, lab media itself can generate H 2 O 2 , and media components interfere with the quantification of H 2 O 2 levels. In this study we describe mechanisms by which media components interfere with H 2 O 2 determinations, and we identify simple ways to minimize and correct for this interference. Using these techniques, it was shown that standard media generate so much H 2 O 2 that most intracellular H 2 O 2 derives from the medium rather than from endogenous metabolism. Indeed, bacteria spread on plates must induce their stress response or else perish. Finally, two straightforward methods were used to sustain low-micromolar steady-state concentrations of H 2 O 2 . In this way we determined that > 2 μM extracellular H 2 O 2 is sufficient to trigger the intracellular OxyR stress response, and > 5 μM begins to impair cell growth in a minimal medium. These concentrations are orders of magnitude lower than the doses that have typically been used in lab experiments. The new approaches should enable workers to study how various organisms cope with natural levels of H 2 O 2 stress. Copyright © 2018 Elsevier Inc. All rights reserved.

Cloning, overexpression, and characterization of cobrotoxin

International Nuclear Information System (INIS)

Hsieh, H.-C.; Kumar, Thallampuranam Krishnaswamy S.; Yu Chin

2004-01-01

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12 mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo autocatalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled ( 15 N and 13 C) and all the resonances ( 1 H, 13 C, and 15 N) in the protein have been unambiguously assigned. 1 H- 15 N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1 H- 15 N chemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the α7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ4-(5α)-dehydrogenase from Rhodococcus jostii RHA1

International Nuclear Information System (INIS)

Oosterwijk, Niels van; Knol, Jan; Dijkhuizen, Lubbert; Geize, Robert van der; Dijkstra, Bauke W.

2011-01-01

The gene for 3-ketosteroid Δ 4 -(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222 1 and diffraction data were collected to a resolution of 1.6 Å. 3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ 4 -(5α)-dehydrogenase [Δ 4 -(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ 4 -(5α)-KSTD enzyme was purified by Ni 2+ –NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C222 1 , with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

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Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao, E-mail: li@sri.org [Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Alabama 35205 (United States)

2005-06-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3{sub 1}21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao

2005-01-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3 1 21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate.

Science.gov (United States)

Nagano, Hiroyuki; Shibano, Kana; Matsumoto, Yu; Yokota, Atsushi; Wada, Masaru

2017-06-01

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.

Phenylalanine ammonia-lyase through evolution: A bioinformatic approach

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Shiva Hemmati

2015-03-01

Full Text Available Phenylalanine ammonia-lyase (PAL is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for phenylketonuric patients in pegylated forms; therefore, screening a PAL with the highest affinity to the substrate is of a great importance. PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species in a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.

Oxidative and Molecular Responses in Capsicum annuum L. after Hydrogen Peroxide, Salicylic Acid and Chitosan Foliar Applications

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Mejía-Teniente, Laura; de Dalia Durán-Flores, Flor; Chapa-Oliver, Angela María; Torres-Pacheco, Irineo; Cruz-Hernández, Andrés; González-Chavira, Mario M.; Ocampo-Velázquez, Rosalía V.; Guevara-González, Ramón G.

2013-01-01

Hydrogen peroxide (H2O2) is an important ROS molecule (Reactive oxygen species) that serves as a signal of oxidative stress and activation of signaling cascades as a result of the early response of the plant to biotic stress. This response can also be generated with the application of elicitors, stable molecules that induce the activation of transduction cascades and hormonal pathways, which trigger induced resistance to environmental stress. In this work, we evaluated the endogenous H2O2 production caused by salicylic acid (SA), chitosan (QN), and H2O2 elicitors in Capsicum annuum L. Hydrogen peroxide production after elicitation, catalase (CAT) and phenylalanine ammonia lyase (PAL) activities, as well as gene expression analysis of cat1, pal, and pathogenesis-related protein 1 (pr1) were determined. Our results displayed that 6.7 and 10 mM SA concentrations, and, 14 and 18 mM H2O2 concentrations, induced an endogenous H2O2 and gene expression. QN treatments induced the same responses in lesser proportion than the other two elicitors. Endogenous H2O2 production monitored during several days, showed results that could be an indicator for determining application opportunity uses in agriculture for maintaining plant alert systems against a stress. PMID:23676352

Optimizing strategy for Escherichia coli growth and hydrogen production during glycerol fermentation in batch culture: Effects of some heavy metal ions and their mixtures

International Nuclear Information System (INIS)

Trchounian, Karen; Poladyan, Anna; Trchounian, Armen

2016-01-01

Highlights: • Effects of heavy metals on Escherichia coli were revealed during glycerol fermentation at pH 6.5. • Ni"2"+ + Fe"2"+ (0.05 mM) mixture stimulated up to 1.5-fold bacterial biomass yield. • Ni"2"+ + Fe"3"+ (0.05 mM), Ni"2"+ + Fe"3"+ + Mo"6"+ (0.02 mM) and Fe"3"+ + Mo"6"+ enhanced (up to 3-fold) H_2 production. • Discrimination between Fe"2"+ and Fe"3"+ was important for H_2 production. • Cu"+ and Cu"2"+ (0.1 mM) inhibited H_2 production. - Abstract: Hydrogen (H_2) is well-known effective, ecologically clean and renewable fuel. Bacterial H_2 production is a promising one and its use in industrial level is expected to increase in the nearest future to establish sustainable and renewable energy source. Escherichia coli wild type BW25113 growth yield was shown to be stimulated 1.3–1.5-fold by nickel (Ni"2"+), iron (Fe"2"+, Fe"3"+) ions and by some metal ion mixtures: Ni"2"+ + Fe"2"+ + molybdenum (Mo"6"+), Ni"2"+ + Fe"3"+, Ni"2"+ + Fe"2"+ and Mo"6"+ + Fe"3"+ in low concentrations (<0.05 mM) stimulated the growth during glycerol (10 g L"−"1) fermentation up to stationary phase at pH 6.5; Ni"2"+ + Fe"2"+ mixture showed the maximal effect. However, the same concentrations of these metals and their mixtures had no effects or slightly inhibited bacterial specific growth rate: it was suppressed ∼1.2-fold upon Ni"2"+, Fe"3"+, Mo"6"+ and Ni"2"+ + Mo"6"+ mixture supplementation. H_2 production by E. coli from glycerol was observed with the yield of 0.75 ± 0.02 mmol L"−"1. Moreover, H_2 yield was markedly stimulated 1.7–3-fold in the presence of Ni"2"+ + Fe"3"+, Ni"2"+ + Fe"3"+ + Mo"6"+ and Fe"3"+ + Mo"6"+ mixtures, but not sole metals: maximal stimulation was established by Fe"3"+ + Mo"6"+ mixture with the concentrations of 0.05 mM and 0.02 mM, respectively. While copper (Cu"+, Cu"2"+) ions in low concentration (0.1 mM) had H_2 production suppressing effect. The results point out that some heavy metal ions and their mixtures can stimulate E

Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

Science.gov (United States)

Chilumuri, Amrutha; Odell, Mark; Milton, Nathaniel G N

2013-11-20

Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

Science.gov (United States)

2013-01-01

Alzheimer’s disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45–50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects. PMID:23968537

Cloning, purification, crystallization and preliminary crystallographic analysis of LsrR from Escherichia coli

International Nuclear Information System (INIS)

Liu, Xiaotian; Wu, Minhao; Sun, Demeng; Zang, Jianye

2010-01-01

The E. coli transcription repressor LsrR has been overexpressed, purified and crystallized. Diffraction data were collected to about 3 Å resolution. In Escherichia coli, the lsr operon is composed of six genes lsrACDBFG which regulate uptake and modification of the signalling molecule AI-2. LsrR is a repressor of the lsr operon and itself, which can bind phospho-AI-2 and be released from the promoter region of the operon and thus activate gene expression. LsrR fused with an HHHHHH sequence at the C-terminus was expressed, purified and crystallized in order to determine its structure and elucidate the molecular mechanism of repression. The crystal belonged to space group I222, with unit-cell parameters a = 79.84, b = 116.65, c = 186.04 Å, and was estimated to contain two protein molecules per asymmetric unit

Engineering Synechocystis PCC6803 for hydrogen production: influence on the tolerance to oxidative and sugar stresses.

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Marcia Ortega-Ramos

Full Text Available In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature. We demonstrate that the hydrogenase-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H₂O₂ and sugar (glucose and glycerol stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase, combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions.

The effects of exogenous catalase on broad-spectrum near-UV (300-400nm) treated Escherichia coli cells

International Nuclear Information System (INIS)

Sammartano, L.J.; Tuveson, R.W.

1984-01-01

Catalase incorporated into plating medium protects against inactivation and mutagenesis by broad-spectrum near-ultraviolet wavelength (300-400nm) (NUV) radiation in strains of Escherichia coli. Plating medium containing catalase does not provide protection against inactivation by wavelengths in the FUV region. Catalase added to the cell suspension during or added immediately after NUV exposure also protects against inactivation. The protection provided by catalase suggests a possible role for hydrogen peroxide in the processes of inactivation and mutagenesis by broad-spectrum NUV. (author)

Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli.

Science.gov (United States)

Li, Qingyan; Fan, Feiyu; Gao, Xiang; Yang, Chen; Bi, Changhao; Tang, Jinlei; Liu, Tao; Zhang, Xueli

2017-11-01

The MEP pathway genes were modulated to investigate whether there were new rate-limiting steps and toxic intermediates in this pathway. Activating IspG led to significant decrease of cell growth and β-carotene production. It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. In addition, intermediate MECPP accumulated in the starting strain, while balanced activation of IspG and IspH could push the carbon flux away from MECPP and led to 73% and 77% increase of β-carotene and lycopene titer respectively. Our work for the first time identified HMBPP to be a cytotoxic intermediate in MEP pathway and demonstrated that balanced activation of IspG and IspH could eliminate accumulation of HMBPP and MECPP and improve isoprenoids production. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

International Nuclear Information System (INIS)

Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

2013-01-01

Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

Overexpression of Enterococcus faecalis elr operon protects from phagocytosis.

Science.gov (United States)

Cortes-Perez, Naima G; Dumoulin, Romain; Gaubert, Stéphane; Lacoux, Caroline; Bugli, Francesca; Martin, Rebeca; Chat, Sophie; Piquand, Kevin; Meylheuc, Thierry; Langella, Philippe; Sanguinetti, Maurizio; Posteraro, Brunella; Rigottier-Gois, Lionel; Serror, Pascale

2015-05-25

Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

Attenuation of cigarette smoke-induced airway mucus production by hydrogen-rich saline in rats.

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Yunye Ning

Full Text Available BACKGROUND: Over-production of mucus is an important pathophysiological feature in chronic airway disease such as chronic obstructive pulmonary disease (COPD and asthma. Cigarette smoking (CS is the leading cause of COPD. Oxidative stress plays a key role in CS-induced airway abnormal mucus production. Hydrogen protected cells and tissues against oxidative damage by scavenging hydroxyl radicals. In the present study we investigated the effect of hydrogen on CS-induced mucus production in rats. METHODS: Male Sprague-Dawley rats were divided into four groups: sham control, CS group, hydrogen-rich saline pretreatment group and hydrogen-rich saline control group. Lung morphology and tissue biochemical changes were determined by immunohistochemistry, Alcian Blue/periodic acid-Schiff staining, TUNEL, western blot and realtime RT-PCR. RESULTS: Hydrogen-rich saline pretreatment attenuated CS-induced mucus accumulation in the bronchiolar lumen, goblet cell hyperplasia, muc5ac over-expression and abnormal cell apoptosis in the airway epithelium as well as malondialdehyde increase in the BALF. The phosphorylation of EGFR at Tyr1068 and Nrf2 up-regulation expression in the rat lungs challenged by CS exposure were also abrogated by hydrogen-rich saline. CONCLUSION: Hydrogen-rich saline pretreatment ameliorated CS-induced airway mucus production and airway epithelium damage in rats. The protective role of hydrogen on CS-exposed rat lungs was achieved at least partly by its free radical scavenging ability. This is the first report to demonstrate that intraperitoneal administration of hydrogen-rich saline protected rat airways against CS damage and it could be promising in treating abnormal airway mucus production in COPD.

Hydrogen sulfide prolongs postharvest storage of fresh-cut pears (Pyrus pyrifolia by alleviation of oxidative damage and inhibition of fungal growth.

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Kang-Di Hu

Full Text Available Hydrogen sulfide (H2S has proved to be a multifunctional signaling molecule in plants and animals. Here, we investigated the role of H2S in the decay of fresh-cut pears (Pyrus pyrifolia. H2S gas released by sodium hydrosulfide (NaHS prolonged the shelf life of fresh-cut pear slices in a dose-dependent manner. Moreover, H2S maintained higher levels of reducing sugar and soluble protein in pear slices. H2S significantly reduced the accumulation of hydrogen peroxide (H2O2, superoxide radicals (•O2(- and malondialdehyde (MDA. Further investigation showed that H2S fumigation up-regulated the activities of antioxidant enzymes ascorbate peroxidase (APX, catalase (CAT, and guaiacol peroxidase (POD, while it down-regulated those of lipoxygenase (LOX, phenylalanine ammonia lyase (PAL and polyphenol oxidase (PPO. Furthermore, H2S fumigation effectively inhibited the growth of two fungal pathogens of pear, Aspergillus niger and Penicillium expansum, suggesting that H2S can be developed as an effective fungicide for postharvest storage. The present study implies that H2S is involved in prolonging postharvest storage of pears by acting as an antioxidant and fungicide.

Overexpression of the Maize Sulfite Oxidase Increases Sulfate and GSH Levels and Enhances Drought Tolerance in Transgenic Tobacco

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Zongliang Xia

2018-03-01

Full Text Available Sulfite oxidase (SO plays a pivotal role in sulfite metabolism. In our previous study, sulfite-oxidizing function of the SO from Zea mays (ZmSO was characterized. To date, the knowledge of ZmSO’s involvement in abiotic stress response is scarce. In this study, we aimed to investigate the role of ZmSO in drought stress. The transcript levels of ZmSO were relatively high in leaves and immature embryos of maize plants, and were up-regulated markedly by PEG-induced water stress. Overexpression of ZmSO improved drought tolerance in tobacco. ZmSO-overexpressing transgenic plants showed higher sulfate and glutathione (GSH levels but lower hydrogen peroxide (H2O2 and malondialdehyde (MDA contents under drought stress, indicating that ZmSO confers drought tolerance by enhancing GSH-dependent antioxidant system that scavenged ROS and reduced membrane injury. In addition, the transgenic plants exhibited more increased stomatal response than the wild-type (WT to water deficit. Interestingly, application of exogenous GSH effectively alleviated growth inhibition in both WT and transgenic plants under drought conditions. qPCR analysis revealed that the expression of several sulfur metabolism-related genes was significantly elevated in the ZmSO-overexpressing lines. Taken together, these results imply that ZmSO confers enhanced drought tolerance in transgenic tobacco plants possibly through affecting stomatal regulation, GSH-dependent antioxidant system, and sulfur metabolism-related gene expression. ZmSO could be exploited for developing drought-tolerant maize varieties in molecular breeding.

Hydrogen sulfide-mediated regulation of contractility in the mouse ileum with electrical stimulation: roles of L-cysteine, cystathionine β-synthase, and K+ channels.

Science.gov (United States)

Yamane, Satoshi; Kanno, Toshio; Nakamura, Hiroyuki; Fujino, Hiromichi; Murayama, Toshihiko

2014-10-05

Hydrogen sulfide (H2S) is considered to be a signaling molecule. The precise mechanisms underlying H2S-related events, including the producing enzymes and target molecules in gastrointestinal tissues, have not been elucidated in detail. We herein examined the involvement of H2S in contractions induced by repeated electrical stimulations (ES). ES-induced contractions were neurotoxin-sensitive and increased by aminooxyacetic acid, an inhibitor of cystathionine β-synthase (CBS) and cystathionine γ-lyase, but not by D,L-propargylglycine, a selective inhibitor of cystathionine γ-lyase, in an ES trial-dependent manner. ES-induced contractions were markedly decreased in the presence of L-cysteine. This response was inhibited by aminooxyacetic acid and an antioxidant, and accelerated by L-methionine, an activator of CBS. The existence of CBS was confirmed. NaHS transiently inhibited ES- and acetylcholine-induced contractions, and sustainably decreased basal tone for at least 20 min after its addition. The treatment with glibenclamide, an ATP-sensitive K+ channel blocker, reduced both the L-cysteine response and NaHS-induced inhibition of contractions. The NaHS-induced decrease in basal tone was inhibited by apamin, a small conductance Ca2+-activated K+ channel blocker. These results suggest that H2S may be endogenously produced via CBS in ES-activated enteric neurons, and regulates contractility via multiple K+ channels in the ileum. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of SeqA and Dam in Escherichia coli gene expression: A global/microarray analysis

DEFF Research Database (Denmark)

Løbner-Olesen, Anders; Marinus, M.G.; Hansen, Flemming G.

2003-01-01

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid...... of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased...... frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status...

Overexpression of NtWRKY50 Increases Resistance to Ralstonia solanacearum and Alters Salicylic Acid and Jasmonic Acid Production in Tobacco.

Science.gov (United States)

Liu, Qiuping; Liu, Ying; Tang, Yuanman; Chen, Juanni; Ding, Wei

2017-01-01

WRKY transcription factors (TFs) modulate plant responses to biotic and abiotic stresses. Here, we characterized a WRKY IIc TF, NtWRKY50, isolated from tobacco ( Nicotiana tabacum ) plants. The results showed that NtWRKY50 is a nuclear-localized protein and that its gene transcript is induced in tobacco when inoculated with the pathogenic bacterium Ralstonia solanacearum . Overexpression of NtWRKY50 enhanced bacterial resistance, which correlated with enhanced SA and JA/ET signaling genes. However, silencing of the NtWRKY50 gene had no obvious effects on plant disease resistance, implying functional redundancy of NtWRKY50 with other TFs. In addition, it was found that NtWRKY50 can be induced by various biotic or abiotic stresses, such as Potato virus Y, Rhizoctonia solani, Phytophthora parasitica , hydrogen peroxide, heat, cold, and wounding as well as the hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Importantly, additional analysis suggests that NtWRKY50 overexpression markedly promotes SA levels but prevents pathogen-induced JA production. These data indicate that NtWRKY50 overexpression leads to altered SA and JA content, increased expression of defense-related genes and enhanced plant resistance to R. solanacearum. These probably due to increased activity of endogenous NtWRKY50 gene or could be gain-of-function phenotypes by altering the profile of genes affected by NtWRKY50 .

Synthesis of d‐ and l‐Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process†

Science.gov (United States)

Parmeggiani, Fabio; Lovelock, Sarah L.; Weise, Nicholas J.; Ahmed, Syed T.

2015-01-01

Abstract The synthesis of substituted d‐phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one‐pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high‐throughput solid‐phase screening method has also been developed to identify PALs with higher rates of formation of non‐natural d‐phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d‐configured product. Furthermore, the system was extended to the preparation of those l‐phenylalanines which are obtained with a low ee value using PAL amination. PMID:27478261

Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process**

Science.gov (United States)

Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

2015-01-01

The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination. PMID:25728350

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

OpenAIRE

Bogani, Federica; Boehmer, Paul E.

2008-01-01

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymeras...

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington’s disease

Science.gov (United States)

Paul, Bindu D.; Sbodio, Juan I.; Xu, Risheng; Vandiver, M. Scott; Cha, Jiyoung Y.; Snowman, Adele M.; Snyder, Solomon H.

2015-01-01

Huntington’s disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes1. Huntington’s disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington’s disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity2. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington’s disease tissues, which may mediate Huntington’s disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington’s disease tissues and in intact mouse models of Huntington’s disease, suggesting therapeutic potential. PMID:24670645

Modular optimization of heterologous pathways for de novo synthesis of (2S-naringenin in Escherichia coli.

Directory of Open Access Journals (Sweden)

Junjun Wu

Full Text Available Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, chorismate mutase/prephenate dehydrogenase (CM/PDH, tyrosine ammonia lyase (TAL, 4-coumarate:CoA ligase (4CL, chalcone synthase (CHS, chalcone isomerase (CHI, malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.

[Overexpression of FKS1 to improve yeast autolysis-stress].

Science.gov (United States)

Li, Jia; Wang, Jinjing; Li, Qi

2015-09-01

With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

Energy Technology Data Exchange (ETDEWEB)

Fon, E.A.; Sarrazin, J.; Rouleau, G.A. [Montreal General Hospital (Canada)] [and others

1995-12-18

Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.

Production of endo-pectate lyase by two stage cultivation of Erwinia carotovora

Energy Technology Data Exchange (ETDEWEB)

Fukuoka, Satoshi; Kobayashi, Yoshiaki

1987-02-26

The productivity of endo-pectate lyase from Erwinia carotovora GIR 1044 was found to be greatly improved by two stage cultivation: in the first stage the bacterium was grown with an inducing carbon source, e.g., pectin, and in the second stage it was cultivated with glycerol, xylose, or fructose with the addition of monosodium L-glutamate as nitrogen source. In the two stage cultivation using pectin or glycerol as the carbon source the enzyme activity reached 400 units/ml, almost 3 times as much as that of one stage cultivation in a 10 liter fermentor. Using two stage cultivation in the 200 liter fermentor improved enzyme productivity over that in the 10 liter fermentor, with 500 units/ml of activity. Compared with the cultivation in Erlenmeyer flasks, fermentor cultivation improved enzyme productivity. The optimum cultivating conditions were agitation of 480 rpm with aeration of 0.5 vvm at 28 /sup 0/C. (4 figs, 4 tabs, 14 refs)

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling.

Science.gov (United States)

Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver

2014-07-01

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates. Copyright © 2014 Elsevier Inc. All rights reserved.

Chronic aerobic exercise training alleviates myocardial fibrosis in aged rats through restoring bioavailability of hydrogen sulfide.

Science.gov (United States)

Ma, Ning; Liu, Hong-Mei; Xia, Ting; Liu, Jian-Dong; Wang, Xiao-Ze

2018-06-02

Age-related fibrosis is attenuated by aerobic exercise; however, little is known concerning the underlying molecular mechanism. To address this question, aged rats were given moderate-intensity exercise for 12 weeks. After exercise in aged rats, hydrogen sulfide (H2S) levels in plasma and heart increased 39.8% and 90.9%, respectively. Exercise upregulated expression of cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) in heart of aged rats. Furthermore, aged rats were given moderate-intensity exercise for 12 weeks or treated with NaHS (intraperitoneal injection of 0.1 ml/kg/day of 0.28 mol/l NaHS). After exercise in aged rats, Masson-trichrome staining area decreased 34.8% and myocardial hydroxyproline levels decreased 29.6%. Exercise downregulated expression of collagen-I and α-SMA in heart of aged rats. Exercise in aged rats reduced malondialdehyde levels in plasma and heart and 3-nitrotyrosine in heart. Exercise in aged rats reduced mRNA and protein expression of CHOP, GRP78, and XBP1. Exercise also reduced mRNA and protein expression of IL-6 and MCP-1 and suppressed activation of JNK in aging heart. Similar effects were demonstrated in aged rats treated with NaHS. Collectively, exercise restored bioavailability of hydrogen sulfide in the heart of aged rats, which partly explained the benefits of exercise against myocardial fibrosis of aged population.

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

Science.gov (United States)

Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

2001-01-01

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

Synthesis of avenanthramides using engineered Escherichia coli.

Science.gov (United States)

Lee, Su Jin; Sim, Geun Young; Kang, Hyunook; Yeo, Won Seok; Kim, Bong-Gyu; Ahn, Joong-Hoon

2018-03-22

Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource. We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D. Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns

Congenital Adrenal Hyperplasia due to 17-alpha-hydoxylase/17,20-lyase Deficiency Presenting with Hypertension and Pseudohermaphroditism: First Case Report from Oman

Directory of Open Access Journals (Sweden)

Waad-Allah S. Mula-Abed

2014-01-01

Full Text Available This is the first report of congenital adrenal hyperplasia (CAH due to combined 17α-hydroxylase/17,20 lyase deficiency in an Omani patient who was initially treated for many years as a case of hypertension. CAH is an uncommon disorder that results from a defect in steroid hormones biosynthesis in the adrenal cortex. The clinical presentation depends on the site of enzymatic mutations and the types of accumulated steroid precursors. A 22-year-old woman who was diagnosed to have hypertension since the age of 10 years who was treated with anti-hypertensive therapy was referred to the National Diabetes and Endocrine Centre, Royal Hospital, Oman. The patient also had primary amenorrhea and features of sexual infantilism. Full laboratory and radio-imaging investigations were done. Adrenal steroids, pituitary function and karyotyping study were performed and the diagnosis was confirmed by molecular mutation study. Laboratory investigations revealed adrenal steroids and pituitary hormones profile in addition to 46XY karyotype that are consistent with the diagnosis of CAH due to 17α-hydroxylase deficiency. Extensive laboratory workup revealed low levels of serum cortisol (and its precursors 17α-hydroxyprogesterone and 11-deoxycortisol, adrenal androgens (dehydroepiandrosterone sulfate and androstenedione, and estrogen (estradiol; and high levels of mineralocorticoids precursors (11-deoxycorticosterone and corticosterone with high levels of ACTH, FSH and LH. Mutation analysis revealed CYP17A1-homozygous mutation (c.287G>A p.Arg96Gln resulting in the complete absence of 17α-hydroxylase/17,20-lyase activity. The patient was treated with dexamethasone and ethinyl estradiol with cessation of anti-hypertensive therapy. A review of the literature was conducted to identify previous studies related to this subtype of CAH. This is the first biochemically and genetically proven case of CAH due to 17α-hydroxylase/17,20-lyase deficiency in Oman and in the Arab

MET overexpression, gene amplification and relevant clinicopathological features in gastric adenocarcinoma.

Science.gov (United States)

Zhang, Jing; Guo, Lei; Liu, Xiuyun; Li, Wenbin; Ying, Jianming

2017-02-07

This study was conducted to investigate the expression of MET in Chinese gastric adenocarcinoma cohort, the correlation between MET overexpression and clinical pathological features, HER2 expression and MET gene amplification. A total of 816 gastric adenocarcinoma patients were included and MET and HER2 immunohistochemical (IHC) staining were performed. IHC and dual-color silver in situ hybridization analysis were performed in the tissue microarrays, constructed from the 240 patients who were randomly selected. MET overexpression (IHC 3+) was observed in 6.0% (49/816) of the cohort. MET overexpression rate was higher in patients with poor prognostic factors, such as clinical stages III/IV (p =0.012) and pathologic stages T3/T4 (p =0.027). The HER2 overexpression (IHC 3+) rate was 8.8% (72/816) and MET overexpression rate was higher in HER2 positive patients (9.7%, 7/72). A high concordance rate (94.6%) between MET overexpression and gene amplification was demonstrated. Therefore, MET overexpression could serve as a prognostic biomarker and a potential therapeutic target for gastric cancer.

Expression, purification and crystallization of the Cmi immunity protein from Escherichia coli

International Nuclear Information System (INIS)

Römer, Christin; Patzer, Silke I.; Albrecht, Reinhard; Zeth, Kornelius; Braun, Volkmar

2011-01-01

The colicin M immunity protein Cmi protects E. coli cells against killing by colicin M. The Cmi protein was produced for structure determination and crystals were obtained which diffracted to high resolution. Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 Å in the orthorhombic space group C222 1 . The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 Å, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%

The Skin Bacterium Propionibacterium acnes Employs Two Variants of Hyaluronate Lyase with Distinct Properties

DEFF Research Database (Denmark)

Nazipi, Seven; Stødkilde-Jørgensen, Kristian; Scavenius, Carsten

2017-01-01

Hyaluronic acid (HA) and other glycosaminoglycans are extracellular matrix components in the human epidermis and dermis. One of the most prevalent skin microorganisms, Propionibacterium acnes, possesses HA-degrading activity, possibly conferred by the enzyme hyaluronate lyase (HYL). In this study......, we identified the HYL of P. acnes and investigated the genotypic and phenotypic characteristics. Investigations include the generation of a P. acneshyl knockout mutant and HYL activity assays to determine the substrate range and formed products. We found that P. acnes employs two distinct variants...... of the observed differences between P. acnes phylotype IA and IB/II strains. Whereas type IA strains are primarily found on the skin surface and associated with acne vulgaris, type IB/II strains are more often associated with soft and deep tissue infections, which would require elaborate tissue invasion...

Hand1 overexpression inhibits medulloblastoma metastasis

Energy Technology Data Exchange (ETDEWEB)

Asuthkar, Swapna; Guda, Maheedhara R. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Martin, Sarah E. [Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Antony, Reuben; Fernandez, Karen [Department of Pediatrics, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Lin, Julian [Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Tsung, Andrew J. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Illinois Neurological Institute, Peoria, IL 61656 (United States); Velpula, Kiran K., E-mail: velpula@uic.edu [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States)

2016-08-19

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

Hand1 overexpression inhibits medulloblastoma metastasis

International Nuclear Information System (INIS)

Asuthkar, Swapna; Guda, Maheedhara R.; Martin, Sarah E.; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J.; Velpula, Kiran K.

2016-01-01

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents.

OpenAIRE

McEvoy, J L; Murata, H; Chatterjee, A K

1990-01-01

recA-mediated production of pectin lyase (PNL) and the bacteriocin carotovoricin occurs in Erwinia carotovora subsp. carotovora 71 when this organism is subjected to agents that damage or inhibit the synthesis of DNA. The structural gene pnlA was isolated from a strain 71 cosmid gene library following mobilization of the cosmids into a moderate PNL producer, strain 193. The cosmid complemented pnl::Tn5 but not ctv::Tn5 mutations. A constitutive level of PNL activity was detected in RecA+ and ...

Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

International Nuclear Information System (INIS)

Iwaki, Jun; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Hasegawa, Tsunemi

2005-01-01

Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K. Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K

Hydrogen system (hydrogen fuels feasibility)

International Nuclear Information System (INIS)

Guarna, S.

1991-07-01

This feasibility study on the production and use of hydrogen fuels for industry and domestic purposes includes the following aspects: physical and chemical properties of hydrogen; production methods steam reforming of natural gas, hydrolysis of water; liquid and gaseous hydrogen transportation and storage (hydrogen-hydride technology); environmental impacts, safety and economics of hydrogen fuel cells for power generation and hydrogen automotive fuels; relevant international research programs

Porphyromonas gingivalis hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation.

Science.gov (United States)

Nakamura, Suguru; Shioya, Koki; Hiraoka, B Yukihiro; Suzuki, Nao; Hoshino, Tomonori; Fujiwara, Taku; Yoshinari, Nobuo; Ansai, Toshihiro; Yoshida, Akihiro

2018-04-01

Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses H2S production from l-cysteine and analysed the role of H2S using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CH3SH) from l-methionine. Therefore, we analysed H2S and CH3SH production by P. gingivalis W83 and a PG0343-deletion mutant (ΔPG0343) with/without l-cysteine and/or l-methionine. The results indicated that CH3SH is produced constitutively irrespective of the presence of l-methionine, while H2S was greatly increased by both P. gingivalis W83 and ΔPG0343 in the presence of l-cysteine. In contrast, CH3SH production by ΔPG0343 was absent irrespective of the presence of l-methionine, and H2S production was eliminated in the absence of l-cysteine. Thus, CH3SH and H2S production involves different substrates, l-methionine or l-cysteine, respectively. Based on these characteristics, we analysed the roles of CH3SH and H2S in abscess formation in mice by P. gingivalis W83 and ΔPG0343. Abscess formation by P. gingivalis W83, but not ΔPG0343, differed significantly in the presence and absence of l-cysteine. In addition, the presence of l-methionine did not affect the size of abscesses generated by P. gingivalis W83 and ΔPG0343. Therefore, we conclude that H2S produced by P. gingivalis does not induce inflammation; however, H2S enhances inflammation caused by CH3SH. Thus, these results suggest the H2S produced by P. gingivalis plays a supportive role in inflammation caused by methionine γ-lyase.

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

Science.gov (United States)

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 . PMID:26306151

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.

Science.gov (United States)

Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinović-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

2011-12-12

FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L⁻¹ of GDH activity (57 mg L⁻¹). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

Functional analysis of a novel hydrogen peroxide resistance gene in Lactobacillus casei strain Shirota.

Science.gov (United States)

Serata, Masaki; Kiwaki, Mayumi; Iino, Tohru

2016-11-01

Lactic acid bacteria have a variety of mechanisms for tolerance to oxygen and reactive oxygen species, and these mechanisms differ among species. Lactobacillus casei strain Shirota grows well under aerobic conditions, indicating that the various systems involved in oxidative stress resistance function in this strain. To elucidate the mechanism of oxidative stress resistance in L. casei strain Shirota, we examined the transcriptome response to oxygen or hydrogen peroxide exposure. We then focused on an uncharacterized gene that was found to be up-regulated by both oxygen and hydrogen peroxide stress; we named the gene hprA1 (hydrogen peroxide resistance gene). This gene is widely distributed among lactobacilli. We investigated the involvement of this gene in oxidative stress resistance, as well as the mechanism of tolerance to hydrogen peroxide. Growth of L. casei MS105, an hprA1-disrupted mutant, was not affected by oxygen stress, whereas the survival rate of MS105 after hydrogen peroxide treatment was markedly reduced compared to that of the wild-type. However, the activity of MS105 in eliminating hydrogen peroxide was similar to that of the wild-type. We cloned hprA1 from L. caseiShirota and purified recombinant HprA1 protein from Escherichia coli. We demonstrated that the recombinant HprA1 protein bound to iron and prevented the formation of a hydroxyl radical in vitro. Thus, HprA1 protein probably contributes to hydrogen peroxide tolerance in L. casei strain Shirota by binding to iron in the cells and preventing the formation of a hydroxyl radical.

Prognostic implication of aquaporin 1 overexpression in resected lung adenocarcinoma.

Science.gov (United States)

Bellezza, Guido; Vannucci, Jacopo; Bianconi, Fortunato; Metro, Giulio; Del Sordo, Rachele; Andolfi, Marco; Ferri, Ivana; Siccu, Paola; Ludovini, Vienna; Puma, Francesco; Sidoni, Angelo; Cagini, Lucio

2017-12-01

Aquaporins (AQPs) are a group of transmembrane water-selective channel proteins thought to play a role in the regulation of water permeability for plasma membranes. Indeed, high AQP levels have been suggested to promote the progression, invasion and metastasis of tumours. Specifically, AQP1 and AQP5 overexpression in lung adenocarcinoma (AC) have been suggested to be involved in molecular mechanisms in lung cancer. The aim of this retrospective cohort single-centre study was to assess both the levels of expression and therein the prognostic significance, regarding outcome of AQP1 and AQP5 in resected AC patients. Patients with histological diagnoses of lung AC submitted to pulmonary resection were included in this cohort study. Tissue microarrays containing cores from 185 ACs were prepared. AQP1 and AQP5 expressions were assessed by immunohistochemistry. Results were scored as either low (Score 0-2) or high (Score 3-9). Clinical data, pathological tumour-node-metastasis staging and follow-up were recorded. Multivariate Cox survival analysis and Fisher's t-test were performed. AQP1 overexpression was detected in 85 (46%) patients, while AQP5 overexpression was observed in 45 (24%) patients. AQP1 did not result being significantly correlated with clinical and pathological parameters, while AQP5 resulted more expressed in AC with mucinous and papillary predominant patterns. Patients with AQP1 overexpression had shorter disease-free survival (P = 0.001) compared with patients without AQP1 overexpression. Multivariate analysis confirmed that AQP1 overexpression was significantly associated with shorter disease-free survival (P = 0.001). Our results evidenced that AQP1 overexpression resulted in a shorter disease-free survival in lung AC patients. Being so, AQP1 overexpression might be an important prognostic marker in lung AC. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

Science.gov (United States)

Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

2015-09-01

Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

Energy Technology Data Exchange (ETDEWEB)

Yu, Suyeun [Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Jang, Yeogil; Paik, Donggi [Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Lee, Eunil, E-mail: eunil@korea.ac.kr [Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Park, Joong-Jean, E-mail: parkjj@korea.ac.kr [Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of)

2015-10-02

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

International Nuclear Information System (INIS)

Yu, Suyeun; Jang, Yeogil; Paik, Donggi; Lee, Eunil; Park, Joong-Jean

2015-01-01

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

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

Energy Technology Data Exchange (ETDEWEB)

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

2007-07-01

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Overproduction, crystallization and preliminary X-ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli

International Nuclear Information System (INIS)

Garces, Fernando; Fernández, Francisco J.; Pérez-Luque, Rosa; Aguilar, Juan; Baldomà, Laura; Coll, Miquel; Badía, Josefa; Vega, M. Cristina

2007-01-01

UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K. UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å 3 Da −1 and a solvent content of 43%

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1

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Oosterwijk, Niels van; Knol, Jan; Dijkhuizen, Lubbert; Geize, Robert van der; Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl [University of Groningen, Nijenborgh 7, 9747 AG Groningen (Netherlands)

2011-10-01

The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222{sub 1} and diffraction data were collected to a resolution of 1.6 Å. 3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase [Δ{sup 4}-(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ{sup 4}-(5α)-KSTD enzyme was purified by Ni{sup 2+}–NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C222{sub 1}, with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å.

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

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Chiu-Shyan Soo

2017-11-01

Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.

Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

NARCIS (Netherlands)

Yu, Hongyou; de Vos, Paul; Ren, Yijin

OBJECTIVE: The hypothesis of the present study is that overexpression of osteoprotegerin (OPG) promotes preosteoblast maturation. MATERIALS AND METHODS: The preosteoblast cell line MC3T3-E1 was transfected with OPG overexpression. OPG expression was confirmed by enzyme-linked immunosorbent assay

Sulfur metabolism in Escherichia coli and related bacteria: facts and fiction.

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Sekowska, A; Kung, H F; Danchin, A

2000-04-01

Living organisms are composed of macromolecules made of hydrogen, carbon, nitrogen, oxygen, phosphorus and sulfur. Much work has been devoted to the metabolism of the first five elements, but much remains to be understood about sulfur metabolism. We review here the situation in Escherichia coli and related bacteria, where more than one hundred genes involved in sulfur metabolism have already been discovered in this organism. Examination of the genome suggests that many more will be found, especially genes involved in regulation, scavenging of sulfur containing molecules and synthesis of coenzymes or prosthetic groups. Furthermore, the involvement of methionine as the universal start of proteins as well as that of its derivative S-adenosylmethionine in a vast variety of cell processes argue in favour of a major importance of sulfur metabolism in all organisms.

Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

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Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

2014-01-09

The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

In vivo overexpression of Emi1 promotes chromosome instability and tumorigenesis.

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Vaidyanathan, S; Cato, K; Tang, L; Pavey, S; Haass, N K; Gabrielli, B G; Duijf, P H G

2016-10-13

Cell cycle genes are often aberrantly expressed in cancer, but how their misexpression drives tumorigenesis mostly remains unclear. From S phase to early mitosis, EMI1 (also known as FBXO5) inhibits the anaphase-promoting complex/cyclosome, which controls cell cycle progression through the sequential degradation of various substrates. By analyzing 7403 human tumor samples, we find that EMI1 overexpression is widespread in solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. To investigate causality, we generated a transgenic mouse model in which we overexpressed Emi1. Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further demonstrate in vitro that even though EMI1 overexpression may cause mitotic arrest and cell death, it also promotes chromosome instability (CIN) following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for CIN and EMI1 overexpression is a stronger marker for CIN than most well-established ones. The fact that Emi1 overexpression promotes CIN and the formation of solid cancers in vivo indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications.

Antibacterial, Prooxidative and Genotoxic Activities of Gallic Acid and its Copper and Iron Complexes against Escherichia coli

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JONATHAN M. BARCELO

2014-12-01

Full Text Available In this study, gallic acid and its complexes with aluminum and iron were investigated for their antibacterial, pro-oxidative, and genotoxic properties at alkaline pH. At 4.0μmol/mL, gallic acid displayed bacteriostatic property while aluminum-gallic acid and iron-gallic acid complexes showed bactericidal property against Escherichia coli ATCC 25922. A higher antibacterial activity was observed in the turbidimetric assay compared to the well-diffusion assay. The metal complexes of gallic acid also generated a higher concentration of malondialdehyde and hydrogen peroxide compared to gallic acid alone at > 0.50µmol/mL. Using the SOS response of the DNA repair-deficient Escherichia coli PQ37, the metal complexes of gallic acid resulted to a significantly higher SOS Induction Factors (ρ<0.01 at ≥0.25μmol/mL. In addition, gallic acid and its metal complexes decrease the cell surface hydrophobicity of E. coli ATCC 25922 in a dose-dependent manner. The present study suggests that the antibacterial property of gallic acid and its metal complexes against Escherichia coli was caused by its pro-oxidative and genotoxic properties. Since metals are involved in the synthesis of the metal complexes of gallic acid, further tests should be conducted to determine their stability and effects to human health.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the variable lymphocyte receptor 2913 ectodomain fused with internalin B

International Nuclear Information System (INIS)

Lee, Ji Yeon; Kim, Hyoun Sook; Baek, In Wha; Back, Jang Mi; Han, Mi Ra; Kong, Sun-Young; Kim, Ji Hyeon; Kirchdoerfer, Robert N.; Kim, Jae-Ouk; Cooper, Max D.; Wilson, Ian A.; Kim, Hyun-Jung; Han, Byung Woo

2012-01-01

The VLR2913 ectodomain fused with internalin B was crystallized and diffraction data were collected to a maximum resolution of 2.04 Å. In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in the recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, the antigen of which is not known, contains the same HVI amino-acid sequence as that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, identification of the antigen for VLR2913 and the main contributing factors for antigen recognition based on a comparison of VLR2913 and VLR RBC36 has been attempted. To initiate and facilitate this structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of internalin B (InlB-VLR2913-ECD). Three amino-acid residues on the concave surface of the LRR modules of InlB-VLR2913-ECD were mutated, considering important residues for hydrogen bonds in the recognition of H-trisaccharide by VLR RBC36. InlB-VLR2913-ECD was overexpressed in Escherichia coli and was crystallized at 295 K using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.04 Å resolution and could be indexed in the tetragonal space group P4 1 2 1 2 (or P4 3 2 1 2), with unit-cell parameters a = 91.12, b = 91.12, c = 62.87 Å. Assuming that one monomer molecule was present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V M ) was 2.75 Å 3 Da −1 and the solvent content was 55.2%. Structural determination of InlB-VLR2913-ECD by molecular replacement is in progress

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

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Sygmund Christoph

2011-12-01

Full Text Available Abstract Background FAD dependent glucose dehydrogenase (GDH currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1. Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

Small Subunits of Serine Palmitoyltransferase (ssSPTs) and Their Physiological Roles

Science.gov (United States)

2014-02-12

Fumonisin B1, whereas AtssSPTa knockdown lines show increased resistance compared to wild type (59). In addition to that, over expression of AtssSPTb...finding, AtssSPTa overexpression showed increased fumonisin B1 sensitivity and conversely AtssSPTa knockdown lines showed resistance. This suggests...2007. Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin - sensitive and accumulate trihydroxy-18:1 long chain base phosphate

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

Science.gov (United States)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-02-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

Molecular cloning, overexpression, purification and crystallographic analysis of a GH43 β-xylosidase from Bacillus licheniformis.

Science.gov (United States)

Diogo, José Alberto; Zanphorlin, Leticia Maria; Sato, Hélia Harumi; Murakami, Mario Tyago; Ruller, Roberto

2015-08-01

β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.

Error-Prone Translesion DNA Synthesis by Escherichia coli DNA Polymerase IV (DinB on Templates Containing 1,2-dihydro-2-oxoadenine

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Masaki Hori

2010-01-01

Full Text Available Escherichia coli DNA polymerase IV (Pol IV is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg, 5-formyluracil (5-fodU, 5-hydroxymethyluracil (5-hmdU, 7,8-dihydro-8-oxoguanine (8-oxodG and 1,2-dihydro-2-oxoadenine (2-oxodA. Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IV's ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:T→G:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:T→G:C mutations in E. coli, while the increase was significantly greater in E. coli overexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:T→G:C mutations.

Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells.

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Onumah, Ogbeyalu E; Jules, George E; Zhao, Yanfeng; Zhou, LiChun; Yang, Hong; Guo, ZhongMao

2009-06-15

Although it is understood that hydrogen peroxide (H(2)O(2)) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H(2)O(2) in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H(2)O(2). The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0 +/- 4.7 h versus 28.6 +/- 0.8 h, pcatalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G(0)/G(1) phase in hCatTg MAECs compared to the wild-type cells (25.0 +/- 0.9 h versus 15.9 +/- 1.4 h, pinhibitors, p21 and p27, which inhibit the Cdk activity required for the G(0)/G(1)- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H(2)O(2) scavenger, suggest that endogenously produced H(2)O(2) mediates MAEC proliferation by fostering the transition from G(0)/G(1) to S phase.

HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

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Hu, Yingchuan; Bandla, Santhoshi; Godfrey, Tony E.; Tan, Dongfeng; Luketich, James D.; Pennathur, Arjun; Qiu, Xing; Hicks, David G.; Peters, Jeffrey; Zhou, Zhongren

2011-01-01

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of BE, 18 cases of low grade dysplasia and 15 cases of high grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and CISH methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by CISH and high density microarrays. We further confirm the similar frequency of HER2 amplification by CISH (18.10%; 21/116) and SNP 6.0 microarrays (16.4%, 19/116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12.1 % (14/116) of esophageal adenocarcinoma and 6.67% (1/15) of HGD. No HER2 amplification or overexpression was identified in BE or LGD. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by CISH than protein overexpression in esophageal adenocarcinoma (18.10% vs 12.9%). A modified two-step model for esophageal adenocarcinoma HER-2 testing is recommend for clinical esophageal adenocarcinoma HER-2 trial. PMID:21460800

Prognostic implication of NQO1 overexpression in hepatocellular carcinoma.

Science.gov (United States)

Lin, Lijuan; Sun, Jie; Tan, Yan; Li, Zhenling; Kong, Fanyong; Shen, Yue; Liu, Chao; Chen, Litian

2017-11-01

To explore the role of NQO1 overexpression for prognostic implication in hepatocellular carcinoma (HCC), NQO1 mRNA levels were detected in HCC fresh tissue samples of HCC and nontumor tissues, respectively. One hundred fifty-six cases of HCC meeting strict follow-up criteria were selected for immunohistochemical staining of NQO1 protein. Correlations between NQO1 overexpression and clinicopathological features of HCC were evaluated using χ 2 tests, survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient 5-year survival was analyzed using Cox proportional hazards analysis. In results, the levels of NQO1 mRNA were significantly up-regulated in 14 fresh tissue samples of HCC. Immunohistochemical analysis showed that the NQO1 expression and overexpression rates were significantly higher in HCC samples compared with either adjacent nontumor tissues or normal liver tissues. NQO1 overexpression correlated to tumor size, venous infiltration and late pTNM stage of HCC. NQO1 overexpression was also related to low disease-free survival and 5-year survival rates. In the late-stage group, disease-free and 5-year survival rates of patients with NQO1 overexpression were significantly lower than those of patients without NQO1 expression. Further analysis using a Cox proportional hazards regression model revealed that NQO1 expression emerged as a significant independent hazard factor for the 5-year survival rate of patients with HCC. Therefore, NQO1 plays an important role in the progression of HCC. NQO1 may potentially be used as an independent biomarker for prognostic evaluation of HCC. Copyright © 2017. Published by Elsevier Inc.

Tobacco, alcohol, and p53 overexpression in early colorectal neoplasia

International Nuclear Information System (INIS)

Terry, Mary Beth; Neugut, Alfred I; Mansukhani, Mahesh; Waye, Jerome; Harpaz, Noam; Hibshoosh, Hanina

2003-01-01

The p53 tumor suppressor gene is commonly mutated in colorectal cancer. While the effect of p53 mutations on colorectal cancer prognosis has been heavily studied, less is known about how epidemiologic risk factors relate to p53 status, particularly in early colorectal neoplasia prior to clinically invasive colorectal cancer (including adenomas, carcinoma in situ (CIS), and intramucosal carcinoma). We examined p53 status, as measured by protein overexpression, in 157 cases with early colorectal neoplasia selected from three New York City colonoscopy clinics. After collecting paraffin-embedded tissue blocks, immunohistochemistry was performed using an anti-p53 monoclonal mouse IgG 2 a [BP53-12-1] antibody. We analyzed whether p53 status was different for risk factors for colorectal neoplasia relative to a polyp-free control group (n = 508). p53 overexpression was found in 10.3%, 21.7%, and 34.9%, of adenomatous polyps, CIS, and intramucosal cases, respectively. Over 90% of the tumors with p53 overexpression were located in the distal colon and rectum. Heavy cigarette smoking (30+ years) was associated with cases not overexpressing p53 (OR = 1.8, 95% CI = 1.1–2.9) but not with those cases overexpressing p53 (OR = 1.0, 95% CI = 0.4–2.6). Heavy beer consumption (8+ bottles per week) was associated with cases overexpressing p53 (OR = 4.0, 95% CI = 1.3–12.0) but not with cases without p53 overexpression (OR = 1.6, 95% CI = 0.7–3.7). Our findings that p53 overexpression in early colorectal neoplasia may be positively associated with alcohol intake and inversely associated with cigarette smoking are consistent with those of several studies of p53 expression and invasive cancer, and suggest that there may be relationships of smoking and alcohol with p53 early in the adenoma to carcinoma sequence

The hydrogen; L'hydrogene

Energy Technology Data Exchange (ETDEWEB)

NONE

2004-07-01

The hydrogen as an energy system represents nowadays a main challenge (in a scientific, economical and environmental point of view). The physical and chemical characteristics of hydrogen are at first given. Then, the challenges of an hydrogen economy are explained. The different possibilities of hydrogen production are described as well as the distribution systems and the different possibilities of hydrogen storage. Several fuel cells are at last presented: PEMFC, DMFC and SOFC. (O.M.)

A QM/MM study of the reaction mechanism of (R)-hydroxynitrile lyases from Arabidopsis thaliana (AtHNL).

Science.gov (United States)

Zhu, Wenyou; Liu, Yongjun; Zhang, Rui

2015-01-01

Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R-selective HNL enzyme containing an α/β-hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1-C2 bond and the rotation of the generated cyanide ion (CN(-)) follow, afterwards, CN(-) abstracts a proton from His236 via Ser81. The C1-C2 bond cleavage and the protonation of CN(-) correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol(-1)), suggesting that both of the two processes contribute a lot to rate-limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1-C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol(-1). The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN(-) is stabilized by the hydroxyl group of Ser81 and the main-chain NH-groups of Ala13 and Phe82. © 2014 Wiley Periodicals, Inc.

The pectin lyase-encoding gene (pnl) family from Glomerella cingulata: characterization of pnlA and its expression in yeast.

Science.gov (United States)

Templeton, M D; Sharrock, K R; Bowen, J K; Crowhurst, R N; Rikkerink, E H

1994-05-03

Oligodeoxyribonucleotide primers were designed from conserved amino acid (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The polymerase chain reaction (PCR) was used with these primers to amplify genomic DNA from the plant pathogenic fungus Glomerella cingulata. Three different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes from Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-encoding gene from a lambda genomic DNA library prepared from G. cingulata. Nucleotide (nt) sequence data revealed that this gene has seven exons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the five introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein was secreted into the culture medium. Significantly higher expression was achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed yeast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1

International Nuclear Information System (INIS)

Akioka, Makoto; Nakano, Hiroaki; Horikiri, Aya; Tsujimoto, Yoshiyuki; Matsui, Hiroshi; Shimizu, Tetsuya; Nakatsu, Toru; Kato, Hiroaki; Watanabe, Kunihiko

2006-01-01

Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out. To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination

Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

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Karthikeyan Thiyagarajan

Full Text Available Phenylalanine Ammonia Lyase (PAL gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum. The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

International Nuclear Information System (INIS)

Malloy, P.J.

1985-01-01

NADP + -specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with ( 32 P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32 P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32 P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32 P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32 P-labeled IDH was isolated from sonicated extracts of the cells. The 32 P-enzyme was carboxylmethylated and digested with trypsin. A single 32 P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32 P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue

Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

Science.gov (United States)

Lindsey, Rebecca L; Garcia-Toledo, L; Fasulo, D; Gladney, L M; Strockbine, N

2017-09-01

Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies. Published by Elsevier B.V.

Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

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Yanlong Wang

Full Text Available We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli, Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology and KEGG (Kyoto encyclopedia of genes and genomes enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species scavenging, membrane proteins and ABC (ATP binding cassette transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.

An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

Science.gov (United States)

Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

2015-02-01

The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume. © 2014 The Society for Applied Microbiology.

Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

Science.gov (United States)

Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

2014-04-25

Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

General applicability of synthetic gene-overexpression for cell-type ratio control via reprogramming.

Science.gov (United States)

Ishimatsu, Kana; Hata, Takashi; Mochizuki, Atsushi; Sekine, Ryoji; Yamamura, Masayuki; Kiga, Daisuke

2014-09-19

Control of the cell-type ratio in multistable systems requires wide-range control of the initial states of cells. Here, using a synthetic circuit in E. coli, we describe the use of a simple gene-overexpression system combined with a bistable toggle switch, for the purposes of enabling the wide-range control of cellular states and thus generating arbitrary cell-type ratios. Theoretically, overexpression induction temporarily alters the bistable system to a monostable system, in which the location of the single steady state of cells can be manipulated over a wide range by regulating the overexpression levels. This induced cellular state becomes the initial state of the basal bistable system upon overexpression cessation, which restores the original bistable system. We experimentally demonstrated that the overexpression induced a monomodal cell distribution, and subsequent overexpression withdrawal generated a bimodal distribution. Furthermore, as designed theoretically, regulating the overexpression levels by adjusting the concentrations of small molecules generated arbitrary cell-type ratios.

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

CERN Document Server

Foulon, V; Croes, K; Waelkens, E

1999-01-01

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

Solvent Isotope-induced Equilibrium Perturbation for Isocitrate Lyase

Science.gov (United States)

Quartararo, Christine E.; Hadi, Timin; Cahill, Sean M.; Blanchard, John S.

2014-01-01

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacteria’s life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage of D2OV = 2.0 ± 0.1 and D2O[V/Kisocitrate] = 2.2 ± 0.3 arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate and succinate prepared in D2O, would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of NMR spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed di-deuterated succinate, protiated glyoxylate, and mono-deuterated isocitrate, with the transient appearance and disappearance of mono-deuterated succinate. A model for the equilibrium perturbation of substrate species, and their time-dependent isotopic composition is presented. PMID:24261638

Insights into the cellular function of YhdE, a nucleotide pyrophosphatase from Escherichia coli.

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Jin Jin

Full Text Available YhdE, a Maf-like protein in Escherichia coli, exhibits nucleotide pyrophosphatase (PPase activity, yet its cellular function remains unknown. Here, we characterized the PPase activity of YhdE on dTTP, UTP and TTP and determined two crystal structures of YhdE, revealing 'closed' and 'open' conformations of an adaptive active site. Our functional studies demonstrated that YhdE retards cell growth by prolonging the lag and log phases, particularly under stress conditions. Morphology studies showed that yhdE-knockout cells transformed the normal rod shape of wild-type cells to a more spherical form, and the cell wall appeared to become more flexible. In contrast, YhdE overexpression resulted in filamentous cells. This study reveals the previously unknown involvement of YhdE in cell growth inhibition under stress conditions, cell-division arrest and cell-shape maintenance, highlighting YhdE's important role in E. coli cell-cycle checkpoints.

21 CFR 866.3255 - Escherichia coli serological reagents.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate▿

OpenAIRE

Wang, X.; Miller, E. N.; Yomano, L. P.; Zhang, X.; Shanmugam, K. T.; Ingram, L. O.

2011-01-01

Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low Km for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced eth...

Glucose-6-phosphate dehydrogenase protects Escherichia coli from tellurite-mediated oxidative stress.

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Juan M Sandoval

Full Text Available The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH, which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH. Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P, suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH, better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.

Constitutive overexpression of muscarinic receptors leads to vagal hyperreactivity.

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Angelo Livolsi

Full Text Available BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS. Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2 and M(3 receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2 receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2 receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2 receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits. This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2 receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that

Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination

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Mickaël Poidevin

2018-02-01

Full Text Available Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such ‘superspreader’ mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

Ensifer meliloti overexpressing Escherichia coli phytase gene ( appA) improves phosphorus (P) acquisition in maize plants

Science.gov (United States)

Sharma, Vikas; Kumar, Ajit; Archana, G.; Kumar, G. Naresh

2016-10-01

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 ( lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti { E. m (pVA1)} increased its phosphatase and phytase activity by ˜9- and ˜50-fold, respectively, compared to the transformants containing empty plasmid as control { E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein { E. m (pVA1)} and { E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both { E. m (pVA1)} and { E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with { E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

DEFF Research Database (Denmark)

Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

2009-01-01

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

International Nuclear Information System (INIS)

Kedzierska, Barbara; Glinkowska, Monika; Iwanicki, Adam; Obuchowski, Michal; Sojka, Piotr; Thomas, Mark S.; Wegrzyn, Grzegorz

2003-01-01

The bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the 'lysis-versus-lysogeny' decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA + or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII

Hydrogen energy

International Nuclear Information System (INIS)

2005-03-01

This book consists of seven chapters, which deals with hydrogen energy with discover and using of hydrogen, Korean plan for hydrogen economy and background, manufacturing technique on hydrogen like classification and hydrogen manufacture by water splitting, hydrogen storage technique with need and method, hydrogen using technique like fuel cell, hydrogen engine, international trend on involving hydrogen economy, technical current for infrastructure such as hydrogen station and price, regulation, standard, prospect and education for hydrogen safety and system. It has an appendix on related organization with hydrogen and fuel cell.

A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

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Yangrae Cho

Full Text Available Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.

PATHOGENIC POTENTIALS OF ESCHERICHIA COLI ISOLATED ...

African Journals Online (AJOL)

Electrolyte and haematological parameters in rabbits infected with pathogenic isolates of Escherichia coli from rural water supplies in Rivers State, Nigeria, where monitored. Rabbits were orally infected with suspension containing 3x107 cfu /ml of Escherichia coli to induce diarrhoea, and the electrolyte (sodium, potassium ...

Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

International Nuclear Information System (INIS)

Haedener, A.; Tamm, Ch.

1987-01-01

Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-[2- 2 H]phenyl-[2- 2 H]alanine was formed from (E) -[2,2'- 2 H 2 ]cinnamic acid and ammonia in 46% yield, whereas L-phenyl-[2- 13 C, 15 N]alanine was obtained from (E)-[2- 13 C]cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged 15 NH 3 was reisolated as 15 NH 4 Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. [2- 2 H]Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with 2 H 2 O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines. (author)

Paraffin as oxygen vector modulates tyrosine phenol lyase production by Citrobacter freundii MTCC 2424.

Science.gov (United States)

Azmi, Wamik; Kumar, Ajay; Dev, Varun

2013-06-01

The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity : application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

NARCIS (Netherlands)

Stijntjes, G.J.; te Koppele, J.M.; Vermeulen, N P

1992-01-01

An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid,

[The effects of TorR protein on initiation of DNA replication in Escherichia coli].

Science.gov (United States)

Yuan, Yao; Jiaxin, Qiao; Jing, Li; Hui, Li; Morigen, Morigen

2015-03-01

The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.

77 FR 9888 - Shiga Toxin-Producing Escherichia coli

Science.gov (United States)

2012-02-21

... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145). This new date..., that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121...

Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

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Cai, Yongping; Lin, Yi

2013-01-01

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris.

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Fukuta, Yasuhisa; Nanda, Samik; Kato, Yasuo; Yurimoto, Hiroya; Sakai, Yasuyoshi; Komeda, Hidenobu; Asano, Yasuhisa

2011-01-01

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.

The effect of aquaporin 5 overexpression on the Ras signaling pathway

International Nuclear Information System (INIS)

Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

2008-01-01

Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells

Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

Science.gov (United States)

Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

1999-01-01

The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

ERBB-2 overexpression as a risk factor for malignant phaeochromocytomas and paraganglinomas.

Science.gov (United States)

Wang, Weiqing; Zhong, Xu; Ye, Lei; Qi, Yan; Su, TingWei; Wei, Qing; Xie, Jing; Jiang, Lei; Jiang, Yiran; Zhou, Weiwei; Cui, Bin; Ning, Guang

2016-06-01

There are currently no good histological or molecular markers to differentiate benign from malignant phaeochromocytomas and paraganglinomas (PPGLs). Our previous cross-sectional study observed that ERBB-2 overexpression was associated with malignant PPGLs. This study aimed to evaluate the predictive value of ERBB-2 overexpression for metastasis in PPGLs in a large population. A total of 262 patients diagnosed as PPGLs in our institution between 2002 and 2012 were included. We analysed ERBB-2 protein expression in the primary PPGL tumours by immunohistochemistry (IHC) and ERBB-2 amplification by fluorescence in situ hybridization (FISH). Direct Sanger sequencing was performed to examine ERBB-2 exon 20 mutations. The occurrence of malignant PPGLs was documented in the follow-up period. Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate the association between ERBB-2 overexpression and metastasis of PPGLs. Twenty-six (9·9%) patients had ERBB-2 overexpression in their primary PPGL tumours, which was significantly associated with ERBB-2 amplification (17/25, 68%). No ERBB-2 mutation was found. At a median follow-up of 4·5 years, a total of 23 malignant PPGLs were documented, including eight (30·8%) patients in the ERBB-2 overexpression group and 15 (6·4%) patients in the ERBB-2-negative group. The incidence rate of metastasis was 5·3 per 100 person-years vs 1·4 per 100 person-years in the ERBB-2 overexpression and ERBB-2-negative groups (P overexpression was associated with decreased metastasis-free survival (P = 0·001, log-rank test). After adjusting for primary tumour size and location, Cox regression analysis revealed that ERBB-2 overexpression was independently associated with risk of malignant PPGLs (HR = 2·78; 95% CI, 1·12-6·90; P = 0·028). Patients harbouring tumours with ERBB-2 overexpression have a significantly higher risk of developing malignant PPGLs. © 2016 John Wiley & Sons Ltd.

Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

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Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

2012-01-01

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

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Rodrigo S Lacruz

Full Text Available We have previously identified amelotin (AMTN as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL and ameloblastin (AMBN was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

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Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

2015-04-27

The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

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Karhumaa Kaisa

2011-07-01

Full Text Available Abstract Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media

Measurement of dissolved hydrogen and hydrogen gas transfer in a hydrogen-producing reactor

Energy Technology Data Exchange (ETDEWEB)

Shizas, I.; Bagley, D.M. [Toronto Univ., ON (Canada). Dept. of Civil Engineering

2004-07-01

This paper presents a simple method to measure dissolved hydrogen concentrations in the laboratory using standard equipment and a series of hydrogen gas transfer tests. The method was validated by measuring hydrogen gas transfer parameters for an anaerobic reactor system that was purged with 10 per cent carbon dioxide and 90 per cent nitrogen using a coarse bubble diffuser stone. Liquid samples from the reactor were injected into vials and hydrogen was allowed to partition between the liquid and gaseous phases. The concentration of dissolved hydrogen was determined by comparing the headspace injections onto a gas chromatograph and a standard curve. The detection limit was 1.0 x 10{sup -5} mol/L of dissolved hydrogen. The gas transfer rate for hydrogen in basal medium and anaerobic digester sludge was used to validate the method. Results were compared with gas transfer models. In addition to monitoring dissolved hydrogen in reactor systems, this method can help improve hydrogen production potential. 1 ref., 4 figs.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

Science.gov (United States)

Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

2016-09-06

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DEK protein overexpression predicts poor prognosis in pancreatic ductal adenocarcinoma.

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Sun, Jie; Bi, Fangfang; Yang, Yang; Zhang, Yuan; Jin, Aihua; Li, Jinzi; Lin, Zhenhua

2017-02-01

DEK, a transcription factor, is involved in mRNA splicing, transcriptional control, cell division and differentiation. Recent studies suggest that DEK overexpression can promote tumorigenesis in a wide range of cancer cell types. However, little is known concerning the status of DEK in pancreatic ductal adenocarcinoma (PDAC). Based on the microarray data from Gene Expression Omnibus (GEO), the expression levels of DEK mRNA in PDAC tissues were significantly higher than levels in the adjacent non-tumor tissues. To explore the clinical features of DEK overexpression in PDAC, 87 PDAC and 52 normal pancreas tissues were selected for immunoenzyme staining of the DEK protein. Localization of the DEK protein was detected in PANC-1 pancreatic cancer cells using immunofluorescence (IF) staining. The correlations between DEK overexpression and the clinical features of PDAC were evaluated using the Chi-squared (χ2) and Fisher's exact tests. The survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazard models. The expression levels of DEK mRNA in PDAC tissues were significantly higher than that in the adjacent non‑tumor tissues. The DEK protein showed a primarily nuclear staining pattern in PDAC. The positive rate of the DEK protein was 52.9% (46/87) in PDAC, which was significantly higher than that in the adjacent normal pancreatic tissues (7.7%, 4/52). DEK overexpression in PDAC was correlated with tumor size, histological grade, tumor‑node‑metastasis (TNM) stage and overall survival (OS) rates. In addition, multivariate analysis demonstrated that DEK overexpression was an independent prognostic factor along with histological grade and TNM stage in patients with PDAC. In conclusion, DEK overexpression is associated with PDAC progression and may be a potential biomarker for poor prognostic evaluation in PDAC.

CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

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Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

2016-04-01

CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. © 2015 International Federation for Cell Biology.

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum

International Nuclear Information System (INIS)

Barranco-Medina, Sergio; López-Jaramillo, Francisco Javier; Bernier-Villamor, Laura; Sevilla, Francisca; Lázaro, Juan-José

2006-01-01

The isolation, purification, crystallization and molecular-replacement solution of mitochondrial type II peroxiredoxin from P. sativum is reported. A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 Å from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 Å, α = 102.90, β = 104.40, γ = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum

Energy Technology Data Exchange (ETDEWEB)

Barranco-Medina, Sergio [Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada (Spain); López-Jaramillo, Francisco Javier, E-mail: fjljara@ugr.es [Instituto de Biotecnología, Campus Fuentenueva, Universidad de Granada, E-18071 Granada (Spain); Bernier-Villamor, Laura [Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada (Spain); Sevilla, Francisca [Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas, E-30080 Murcia (Spain); Lázaro, Juan-José [Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada (Spain)

2006-07-01

The isolation, purification, crystallization and molecular-replacement solution of mitochondrial type II peroxiredoxin from P. sativum is reported. A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 Å from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 Å, α = 102.90, β = 104.40, γ = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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Vinciane Régnier

Full Text Available The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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Régnier, Vinciane; Billard, Jean-Marie; Gupta, Sapna; Potier, Brigitte; Woerner, Stéphanie; Paly, Evelyne; Ledru, Aurélie; David, Sabrina; Luilier, Sabrina; Bizot, Jean-Charles; Vacano, Guido; Kraus, Jan P; Patterson, David; Kruger, Warren D; Delabar, Jean M; London, Jaqueline

2012-01-01

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes. Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

WDR62 overexpression is associated with a poor prognosis in patients with lung adenocarcinoma.

Science.gov (United States)

Shinmura, Kazuya; Kato, Hisami; Kawanishi, Yuichi; Igarashi, Hisaki; Inoue, Yusuke; Yoshimura, Katsuhiro; Nakamura, Satoki; Fujita, Hidehiko; Funai, Kazuhito; Tanahashi, Masayuki; Niwa, Hiroshi; Ogawa, Hiroshi; Sugimura, Haruhiko

2017-08-01

Human WDR62, which is localized in the cytoplasm including the centrosome, is known to be responsible for primary microcephaly; however, the role of WDR62 abnormality in cancers remains largely unknown. In this study, we aimed to reveal the pathological role of WDR62 abnormality in lung adenocarcinoma (LAC). We first examined the WDR62 mRNA expression level of LAC (n = 64) using a QRT-PCR analysis and found that WDR62 mRNA transcripts were significantly overexpressed in LAC (P = 0.0432, Wilcoxon matched pairs test). An immunohistochemical analysis for LAC (n = 237) showed that WDR62 proteins were also significantly overexpressed in LAC (P overexpression have a short overall survival (P = 0.0378, log-rank test), and a multivariate analysis revealed that WDR62 overexpression was an independent predictor of a poor survival outcome among LAC patients (hazard ratio, 2.032; 95% confidence interval, 1.071-3.777; P = 0.0305). Next, we examined the functional effect of WDR62 overexpression on the lung cancer cell line H1299. WDR62-overexpressing lung cancer cells exhibited an increase in cell growth. Moreover, the concurrent overexpression of WDR62 and TPX2, a WDR62-interacting protein that is also overexpressed in LAC, induced centrosome amplification in the lung cells. Finally, we disclosed that the concurrent overexpression of WDR62 and TPX2 is common in diverse human cancers, using data from the Cancer Genome Atlas. These results suggested that WDR62 overexpression is associated with a poor prognosis in patients with LAC and leads to an increase in the malignant potential of lung cells. © 2017 Wiley Periodicals, Inc.

Overexpression of Myo1e in mouse podocytes enhances cellular endocytosis, migration, and adhesion.

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Jin, Xia; Wang, Wenjing; Mao, Jianhua; Shen, Huijun; Fu, Haidong; Wang, Xia; Gu, Weizhong; Liu, Aimin; Yu, Huimin; Shu, Qiang; Du, Lizhong

2014-02-01

Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte-specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC-transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside-induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e-overexpressing podocytes in the transwell migration assay. Internalization of FITC-transferrin also increased in Myo1e-overexpressing podocytes relative to control cells. Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. © 2013 Wiley Periodicals, Inc.

Energetic heavy ions overcome tumor radioresistance caused by overexpression of Bcl-2

International Nuclear Information System (INIS)

Hamada, Nobuyuki; Hara, Takamitsu; Omura-Minamisawa, Motoko; Funayama, Tomoo; Sakashita, Tetsuya; Sora, Sakura; Yokota, Yuichiro; Nakano, Takashi

2008-01-01

Background and purpose: Overexpression of Bcl-2 is frequent in human cancers and has been associated with radioresistance. Here we investigated the potential impact of heavy ions on Bcl-2 overexpressing tumors. Materials and methods: Bcl-2 cells (Bcl-2 overexpressing HeLa cells) and Neo cells (neomycin resistant gene-expressing HeLa cells) exposed to γ-rays or heavy ions were assessed for the clonogenic survival, apoptosis and cell cycle distribution. Results: Whereas Bcl-2 cells were more resistant to γ-rays (0.2 keV/μm) and helium ions (16.2 keV/μm) than Neo cells, heavy ions (76.3-1610 keV/μm) yielded similar survival regardless of Bcl-2 overexpression. Carbon ions (108 keV/μm) decreased the difference in the apoptotic incidence between Bcl-2 and Neo cells, and prolonged G 2 /M arrest that occurred more extensively in Bcl-2 cells than in Neo cells. Conclusions: High-LET heavy ions overcome tumor radioresistance caused by Bcl-2 overexpression, which may be explained at least in part by the enhanced apoptotic response and prolonged G 2 /M arrest. Thus, heavy-ion therapy may be a promising modality for Bcl-2 overexpressing radioresistant tumors

One Year Experience of Pheburane(®) (Sodium Phenylbutyrate) Treatment in a Patient with Argininosuccinate Lyase Deficiency.

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Uçar, Sema Kalkan; Ozbaran, Burcu; Altinok, Yasemin Atik; Kose, Melis; Canda, Ebru; Kagnici, Mehtap; Coker, Mahmut

2015-01-01

Argininosuccinate lyase deficiency (ASLD) is a urea cycle disorder (UCD) treated with dietary adjustment and nitrogen scavenging agents. "Pheburane(®)" is a new tasteless and odour-free formulation of sodium phenylbutyrate, indicated in the treatment of UCD.A male patient diagnosed with ASLD was put on treatment with the new formulation of sodium phenylbutyrate (granules) for a period of one year, at 500 mg/kg orally in 3 intakes/day. Plasma glutamine, arginine, citrulline, argininosuccinate, serum sodium, potassium, liver function tests and urine orotate all remained unchanged over this period. There was no difference in mean ammonia levels before and after treatment, and no hyperammonemia episode occurred during treatment with Pheburane(®). An improvement in a measurement of quality of life (QOL) was noted after treatment with Pheburane(®). Good metabolic control and improved QOL were achieved throughout the treatment period.

A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum

International Nuclear Information System (INIS)

Sharma, Shailendra Kumar; Modak, Rahul; Sharma, Shilpi; Sharma, Alok Kumar; Sarma, Siddhartha P.; Surolia, Avadhesha; Surolia, Namita

2005-01-01

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

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Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young-Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye-Yeon

2011-01-01

The crystallization of the OmpA periplasmic domain from A. baumannii is described. Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2 1 , with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å 3 Da −1

Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence.

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Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Enooku, Kenichiro; Sato, Masaya; Saigusa, Daisuke; Aoki, Junken; Ishizawa, Takeaki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

2016-01-01

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

International Nuclear Information System (INIS)

Gopalan, Vinod; Islam, Farhadul; Pillai, Suja; Tang, Johnny Cheuk-On; Tong, Daniel King-Hung; Law, Simon; Chan, Kwok-Wah; Lam, Alfred King-Yin

2016-01-01

Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Gopalan, Vinod; Islam, Farhadul; Pillai, Suja [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia); Tang, Johnny Cheuk-On [State Key Laboratory of Chirosciences, Lo Ka Chung Centre for Natural Anti-cancer Drug Development, Department of Applied Biology and Chemical Technology, the Hong Kong Polytechnic University (Hong Kong); Tong, Daniel King-Hung; Law, Simon [Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Chan, Kwok-Wah [Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Lam, Alfred King-Yin, E-mail: a.lam@griffith.edu.au [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia)

2016-11-01

Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

[Effect of luxS overexpression on biofilm formation by Streptococcus mutans].

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He, Zhiyan; Wang, Yuxia; Huang, Zhengwei

2015-09-01

To evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans (Sm). In order to construct pIB-luxS plasmid, the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests. The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot. The growth rate of both Sm wild type strain and its luxS overexpression strain were observed. Methyl thiazolyl tetrazolium (MTT) assay method was used to compare the biofilm formation quantification by both strains at different time points and containing different sucrose. The structures of the biofilms were observed by using confocal laser scanning microscopy, and biofilm-related gene expressions were investigated by real-time PCR. All experiments were performed in triplicate. The luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting. The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference, but biofilm formed by Sm overexpression strain was 0.400 ± 0.009 and 0.609 ± 0.041 at 14 and 24 h, higher than the wild type strain biofilm at the same time point (0.352 ± 0.028 and 0.533 ± 0.014, respectively, P overexpression strain raised to 1.041 ± 0.038, higher than that by the wild type strain (0.831 ± 0.020, P overexpression strain aggregated into distinct clusters on structure, genes expression including gtfB, ftf, gbpB, relA, brpA, smu630, comDE, vicR were increased (6.10 ± 0.12, 3.34 ± 0.07, 8.75 ± 0.13, 2.96 ± 0.04, 5.20 ± 0.19, 2.20 ± 0.06, 2.32 ± 0.07 and 10.67 ± 0.57 fold) compared to the wild-type strain (P < 0.05). Quorum sensing luxS gene can promote the biofilm formation of Sm.

Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. carotovora in response to DNA-damaging agents.

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Liu, Y; Chatterjee, A; Chatterjee, A K

1994-12-01

In most soft-rotting Erwinia spp., including E. carotovora subsp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (Pnl) is activated by DNA-damaging agents such as mitomycin C (MC). Induction of Pnl production in Ecc71 requires a functional recA gene and the rdg locus. DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressors of lambdoid phages, specially phi 80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix-turn-helix motifs. By replacing the rdgB promoter with the IPTG-inducible tac promoter, we have determined that rdgB by itself can activate Pnl production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnlA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of Pnl production, are expressed differently in Ecc71.

Short-Term Vitamin B-6 Restriction Does Not Affect Plasma Concentrations of Hydrogen Sulfide Biomarkers Lanthionine and Homolanthionine in Healthy Men and Women.

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DeRatt, Barbara N; Ralat, Maria A; Gregory, Jesse F

2016-03-09

Suboptimal vitamin B-6 status is associated with increased cardiovascular disease risk, although the mechanism is unknown. The synthesis of the vasodilator hydrogen sulfide occurs through side reactions of the transsulfuration enzymes cystathionine β-synthase and cystathionine γ-lyase, with pyridoxal 5'-phosphate as a coenzyme. Two proposed hydrogen sulfide biomarkers, lanthionine and homolanthionine, are produced concurrently. To determine whether hydrogen sulfide production is reduced by vitamin B-6 deficiency, we examined the relations between plasma concentrations of lanthionine and homolanthionine, along with other components of the transsulfuration pathway (homocysteine, cystathionine, and Cys), in a secondary analysis of samples from 2 vitamin B-6 restriction studies in healthy men and women. Metabolite concentrations were measured in plasma from 23 healthy adults (12 men and 11 women) before and after 28-d controlled dietary vitamin B-6 restriction (0.37 ± 0.04 mg/d). Vitamin B-6 restriction effects on lanthionine and homolanthionine concentrations were assessed. Associations between hydrogen sulfide biomarkers, transsulfuration metabolites, and functional indicators of vitamin B-6 deficiency were analyzed by linear regression. Preprandial plasma lanthionine and homolanthionine concentrations ranged from 89.0 to 372 nmol/L and 5.75 to 32.3 nmol/L, respectively, in healthy adults. Mean lanthionine and homolanthionine concentrations were not affected by vitamin B-6 restriction (P restriction, homolanthionine was positively associated with functional indicators of vitamin B-6 deficiency, which differed from hypothesized negative associations. Plasma lanthionine was positively correlated with the concentration of its precursor, Cys, before (R 2 = 0.36; P = 0.002) and after (R 2 = 0.37; P = 0.002) restriction. Likewise, homolanthionine concentration was positively correlated with its precursor homocysteine, but only in vitamin B-6 adequacy (R 2 = 0.41; P

Dependence of hydrogen-induced lattice defects and hydrogen embrittlement of cold-drawn pearlitic steels on hydrogen trap state, temperature, strain rate and hydrogen content

International Nuclear Information System (INIS)

Doshida, Tomoki; Takai, Kenichi

2014-01-01

The effects of the hydrogen state, temperature, strain rate and hydrogen content on hydrogen embrittlement susceptibility and hydrogen-induced lattice defects were evaluated for cold-drawn pearlitic steel that absorbed hydrogen in two trapping states. Firstly, tensile tests were carried out under various conditions to evaluate hydrogen embrittlement susceptibility. The results showed that peak 2 hydrogen, desorbed at temperatures above 200 °C as determined by thermal desorption analysis (TDA), had no significant effect on hydrogen embrittlement susceptibility. In contrast, hydrogen embrittlement susceptibility increased in the presence of peak 1 hydrogen, desorbed from room temperature to 200 °C as determined by TDA, at temperatures higher than −30 °C, at lower strain rates and with higher hydrogen content. Next, the same effects on hydrogen-induced lattice defects were also evaluated by TDA using hydrogen as a probe. Peak 2 hydrogen showed no significant effect on either hydrogen-induced lattice defects or hydrogen embrittlement susceptibility. It was found that hydrogen-induced lattice defects formed under the conditions where hydrogen embrittlement susceptibility increased. This relationship indicates that hydrogen embrittlement susceptibility was higher under the conditions where the formation of hydrogen-induced lattice defects tended to be enhanced. Since hydrogen-induced lattice defects formed by the interaction between hydrogen and strain were annihilated by annealing at a temperature of 200 °C, they were presumably vacancies or vacancy clusters. One of the common atomic-level changes that occur in cold-drawn pearlitic steel showing higher hydrogen embrittlement susceptibility is the formation of vacancies and vacancy clusters

Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

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Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

2008-01-01

tRNA (m 7 G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m 7 G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7 -methylguanosine (m 7 G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His 6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2 1

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans.

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Rani, Aruna; Dhillon, Arun; Sharma, Kedar; Goyal, Arun

2018-04-01

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α) 6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×10 5 ) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

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Sajadi, Elaheh; Babaipour, Valiollah; Deldar, Ali Asghar; Yakhchali, Bagher; Fatemi, Seyed Safa-Ali

2017-09-01

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

A new Em-like protein from Lactuca sativa, LsEm1, enhances drought and salt stress tolerance in Escherichia coli and rice.

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Xiang, Dian-Jun; Man, Li-Li; Zhang, Chun-Lan; Peng-Liu; Li, Zhi-Gang; Zheng, Gen-Chang

2018-02-07

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these

Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

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Xue, L.

2018-01-01

Full Text Available Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil. Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids solubilizing phosphate. Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can solubilize P in tricalcium phosphate (TCP medium. We aimed to obtain the gene expressed in Escherichia coli, which can enhance organic acid secretion. Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5'-end of RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid secretion ability of the transformed E. coli DH5α™. Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of 233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg·l-1 acetic acid within 48 h. The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™ clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert. Conclusions. We obtained a

Hydrogen sensor

Science.gov (United States)

Duan, Yixiang; Jia, Quanxi; Cao, Wenqing

2010-11-23

A hydrogen sensor for detecting/quantitating hydrogen and hydrogen isotopes includes a sampling line and a microplasma generator that excites hydrogen from a gas sample and produces light emission from excited hydrogen. A power supply provides power to the microplasma generator, and a spectrometer generates an emission spectrum from the light emission. A programmable computer is adapted for determining whether or not the gas sample includes hydrogen, and for quantitating the amount of hydrogen and/or hydrogen isotopes are present in the gas sample.

Immunohistochemical determination of HER-2/neu overexpression in malignant melanoma reveals no prognostic value, while c-Kit (CD117 overexpression exhibits potential therapeutic implications

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Potti Anil

2003-01-01

Full Text Available Abstract Background HER-2/neu and c-kit (CD117 onco-protein are increasingly being recognized as targets for therapy in solid tumors, but data on their role in malignant melanoma is currently limited. We studied the prevalence of overexpression of HER-2/neu and c-Kit in 202 patients with malignant melanoma to evaluate a possible prognostic value of these molecular targets in malignant melanoma. Methods Overexpression of HER-2/neu and c-Kit was evaluated using immunohistochemical assays in 202 archival tissue specimens. Results Between 1991 and 2001, 202 subjects (109 males; 54% and 93 females; 46% with malignant melanoma were studied with a mean age of 57 years (age range: 15–101 years. The most common histologic type was amelanotic melanoma (n = 62; 30.7% followed by superficial spreading melanoma (n = 54; 26.7%. The depth of penetration of melanoma (Breslow thickness, pT Stage ranged from 0.4 mm (stage pT1 to 8.0 mm (stage pT4A. Mean thickness was 2.6 mm (stage pT3A. The ECOG performance scores ranged from 0 to 3. Only 2 patients (0.9% revealed HER-2/neu overexpression, whereas 46 (22.8% revealed c-Kit overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit positive and negative groups (p = 0.36. Interestingly, not only was c-Kit more likely to be overexpressed in the superficial spreading type, a preliminary association between the presence or absence of c-Kit overexpression and the existence of another second primary tumor was also observed. Conclusions The results of our large study indicate that the HER-2/neu onco-protein neither has a role in melanogenesis nor is a potential target for clinical trials with monoclonal antibody therapy. This indicates there is no role for its testing in patients with malignant melanoma. Although c-Kit, expressed preferentially in the superficial spreading type, may not have prognostic value, it does have significant therapeutic implications as a

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar ‘switch complex’ in Vibrio cholerae

International Nuclear Information System (INIS)

Khamrui, Susmita; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

2010-01-01

A chemotaxis response regulator CheY3 from V. cholerae has been cloned, overexpressed, purified and crystallized. The crystals of CheY3 diffracted to 1.86 Å resolution. Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1–CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ‘switch complex’ in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni–NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 Å 3 Da −1 (47% solvent content) assuming the presence of one molecule per asymmetric unit

Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

Science.gov (United States)

Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro

2016-11-01

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Clinicopathological correlation and prognostic significance of sonic hedgehog protein overexpression in human gastric cancer.

Science.gov (United States)

Niu, Yanyang; Li, Fang; Tang, Bo; Shi, Yan; Hao, Yingxue; Yu, Peiwu

2014-01-01

This study investigated the expression of Sonic Hedgehog (Shh) protein in gastric cancer, and correlated it with clinicopathological parameters. The prognostic significance of Shh protein was analyzed. Shh protein expression was evaluated in 113 cases of gastric cancer and 60 cases of normal gastric mucosa. The immunoreactivity was scored semi quantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely non-overexpression group with score 0 or 1, and overexpression group with score 2 or 3. The overexpression of Shh protein was correlated with clinicopathological parameters. Survival analysis was then performed to determine the Shh protein prognostic significance in gastric cancer. In immunohistochemistry study, nineteen (31.7%) normal gastric mucosa revealed Shh protein overexpression, while eighty-one (71.7%) gastric cancer revealed overexpression. The expression of Shh protein were significantly higher in gastric cancer tissues than in normal gastric mucosa (P overexpression and non-expression groups P = 0.168 and 0.071). However, Shh overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 1.187, P = 0.041). Shh protein expression is upregulated and is statistically correlated with age, tumor differentiation, depth of invasion, pathologic staging, and nodal metastasis. The Shh protein overexpression is a significant independent prognostic factor in multivariate Cox regression analysis in gastric cancer.

Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

Energy Technology Data Exchange (ETDEWEB)

Haedener, A.; Tamm, Ch.

1987-11-01

Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-(2-/sup 2/H)phenyl-(2-/sup 2/H)alanine was formed from (E) -(2,2'-/sup 2/H/sub 2/)cinnamic acid and ammonia in 46% yield, whereas L-phenyl-(2-/sup 13/C, /sup 15/N)alanine was obtained from (E)-(2-/sup 13/C)cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged /sup 15/NH/sub 3/ was reisolated as /sup 15/NH/sub 4/Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. (2-/sup 2/H)Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with /sup 2/H/sub 2/O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines.

Escherichia Coli

Science.gov (United States)

Goodsell, David S.

2009-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

Stabilization of Phenylalanine Ammonia Lyase from Rhodotorula glutinis by Encapsulation in Polyethyleneimine-Mediated Biomimetic Silica.

Science.gov (United States)

Cui, Jiandong; Liang, Longhao; Han, Cong; Lin Liu, Rong

2015-06-01

Phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis was encapsulated within polyethyleneimine-mediated biomimetic silica. The main factors in the preparation of biomimetic silica were optimized by response surface methodology (RSM). Compared to free PAL (about 2 U), the encapsulated PAL retained more than 43 % of their initial activity after 1 h of incubation time at 60 °C, whereas free PAL lost most of activity in the same conditions. It was clearly indicated that the thermal stability of PAL was improved by encapsulation. Moreover, the encapsulated PAL exhibited the excellent stability of the enzyme against denaturants and storage stability, and pH stability was improved by encapsulation. Operational stability of 7 reaction cycles showed that the encapsulated PAL was stable. Nevertheless, the K m value of encapsulated PAL in biomimetic silica was higher than that of the free PAL due to lower total surface area and increased mass transfer resistance.

Hail hydrogen

International Nuclear Information System (INIS)

Hairston, D.

1996-01-01

After years of being scorned and maligned, hydrogen is finding favor in environmental and process applications. There is enormous demand for the industrial gas from petroleum refiners, who need in creasing amounts of hydrogen to remove sulfur and other contaminants from crude oil. In pulp and paper mills, hydrogen is turning up as hydrogen peroxide, displacing bleaching agents based on chlorine. Now, new technologies for making hydrogen have the industry abuzz. With better capabilities of being generated onsite at higher purity levels, recycled and reused, hydrogen is being prepped for a range of applications, from waste reduction to purification of Nylon 6 and hydrogenation of specialty chemicals. The paper discusses the strong market demand for hydrogen, easier routes being developed for hydrogen production, and the use of hydrogen in the future

Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

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Márcia Faria

2016-01-01

Full Text Available RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs and 33 follicular thyroid adenomas (FTAs. RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01 and poorer clinical outcome (P = 0.01 suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions.

Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

Science.gov (United States)

Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

2016-01-01

RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

Correlation between human papillomavirus and p16 overexpression in oropharyngeal tumours

DEFF Research Database (Denmark)

Grønhøj Larsen, C; Gyldenløve, M; Jensen, D H

2014-01-01

A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diag...

Transgenic overexpression of adenine nucleotide translocase 1 protects ischemic hearts against oxidative stress.

Science.gov (United States)

Klumpe, Inga; Savvatis, Konstantinos; Westermann, Dirk; Tschöpe, Carsten; Rauch, Ursula; Landmesser, Ulf; Schultheiss, Heinz-Peter; Dörner, Andrea

2016-06-01

Ischemia impairs the adenine nucleotide translocase (ANT), which transports ADP and ATP across the inner mitochondrial membrane. We investigated whether ANT1 overexpression has protective effects on ischemic hearts. Myocardial infarction was induced in wild-type (WT) and heart-specific ANT1-transgenic (ANT1-TG) rats, and hypoxia was set in isolated cardiomyocytes. ANT1 overexpression reduced the myocardial infarct area and increased the survival rate of infarcted rats. Reduced ANT1 expression and increased 4-hydroxynonenal modification of ANT paralleled to impaired ANT function in infarcted WT hearts. ANT1 overexpression improved ANT expression and function. This was accompanied by reduced mitochondrial cytochrome C release and caspase-3 activation. ANT1-TG hearts suffered less from oxidative stress, as shown by lower protein carbonylation and 4-hydroxynonenal modification of ANT. ANT1 overexpression also increased cell survival of hypoxic cardiomyocytes and attenuated reactive oxygen species (ROS) production. This was linked to higher stability of mitochondrial membrane potential and lower activity of ROS detoxifying catalase. ANT1-TG cardiomyocytes also showed higher resistance against H2O2 treatment, which was independent of catalase activity. In conclusion, ANT1 overexpression compensates impaired ANT activity under oxygen-restricted conditions. It reduces ROS production and oxidative stress, stabilizes mitochondrial integrity, and increases survival, making ANT1 a component in ROS management and heart protection during ischemia. ANT1 overexpression reduces infarct size and increases survival after infarction. ANT1 overexpression compensates restricted ANT expression and function in infarcted hearts. Increased ANT1 expression enhances mitochondrial integrity. ANT1-overexpressing hearts reduce oxidative stress by decreasing ROS generation. ANT1 is a component in ROS management and heart protection.

Overexpression of K-p21Ras play a prominent role in lung cancer

Science.gov (United States)

Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

International Nuclear Information System (INIS)

Donnelly, C.E.; Walker, G.C.

1989-01-01

Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus

Short-Term Vitamin B-6 Restriction Does Not Affect Plasma Concentrations of Hydrogen Sulfide Biomarkers Lanthionine and Homolanthionine in Healthy Men and Women123

Science.gov (United States)

DeRatt, Barbara N; Ralat, Maria A; Gregory, Jesse F

2016-01-01

Background: Suboptimal vitamin B-6 status is associated with increased cardiovascular disease risk, although the mechanism is unknown. The synthesis of the vasodilator hydrogen sulfide occurs through side reactions of the transsulfuration enzymes cystathionine β-synthase and cystathionine γ-lyase, with pyridoxal 5′-phosphate as a coenzyme. Two proposed hydrogen sulfide biomarkers, lanthionine and homolanthionine, are produced concurrently. Objective: To determine whether hydrogen sulfide production is reduced by vitamin B-6 deficiency, we examined the relations between plasma concentrations of lanthionine and homolanthionine, along with other components of the transsulfuration pathway (homocysteine, cystathionine, and Cys), in a secondary analysis of samples from 2 vitamin B-6 restriction studies in healthy men and women. Methods: Metabolite concentrations were measured in plasma from 23 healthy adults (12 men and 11 women) before and after 28-d controlled dietary vitamin B-6 restriction (0.37 ± 0.04 mg/d). Vitamin B-6 restriction effects on lanthionine and homolanthionine concentrations were assessed. Associations between hydrogen sulfide biomarkers, transsulfuration metabolites, and functional indicators of vitamin B-6 deficiency were analyzed by linear regression. Results: Preprandial plasma lanthionine and homolanthionine concentrations ranged from 89.0 to 372 nmol/L and 5.75 to 32.3 nmol/L, respectively, in healthy adults. Mean lanthionine and homolanthionine concentrations were not affected by vitamin B-6 restriction (P restriction, homolanthionine was positively associated with functional indicators of vitamin B-6 deficiency, which differed from hypothesized negative associations. Plasma lanthionine was positively correlated with the concentration of its precursor, Cys, before (R2 = 0.36; P = 0.002) and after (R2 = 0.37; P = 0.002) restriction. Likewise, homolanthionine concentration was positively correlated with its precursor homocysteine, but only in

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

Science.gov (United States)

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

S1P Lyase Regulation of Thymic Egress and Oncogenic Inflammatory Signaling

Science.gov (United States)

Kumar, Ashok; Zamora-Pineda, Jesus; Degagné, Emilie

2017-01-01

Sphingosine-1-phosphate (S1P) is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL). SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC). Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1, the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes. PMID:29333002

PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation.

Science.gov (United States)

Jiang, Jingjing; Yao, Lina; Yu, Youjian; Liang, Ying; Jiang, Jianxia; Ye, Nenghui; Miao, Ying; Cao, Jiashu

2014-12-01

Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

S1P Lyase Regulation of Thymic Egress and Oncogenic Inflammatory Signaling

Directory of Open Access Journals (Sweden)

Ashok Kumar

2017-01-01

Full Text Available Sphingosine-1-phosphate (S1P is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL. SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC. Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1, the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes.

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

International Nuclear Information System (INIS)

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

2007-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2007-04-01

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

Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

Directory of Open Access Journals (Sweden)

Qing Jin

Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

Hydrogen molecules and hydrogen-related defects in crystalline silicon

Science.gov (United States)

Fukata, N.; Sasaki, S.; Murakami, K.; Ishioka, K.; Nakamura, K. G.; Kitajima, M.; Fujimura, S.; Kikuchi, J.; Haneda, H.

1997-09-01

We have found that hydrogen exists in molecular form in crystalline silicon treated with hydrogen atoms in the downstream of a hydrogen plasma. The vibrational Raman line of hydrogen molecules is observed at 4158 cm-1 for silicon samples hydrogenated between 180 and 500 °C. The assignment of the Raman line is confirmed by its isotope shift to 2990 cm-1 for silicon treated with deuterium atoms. The Raman intensity has a maximum for hydrogenation at 400 °C. The vibrational Raman line of the hydrogen molecules is broad and asymmetric. It consists of at least two components, possibly arising from hydrogen molecules in different occupation sites in crystalline silicon. The rotational Raman line of hydrogen molecules is observed at 590 cm-1. The Raman band of Si-H stretching is observed for hydrogenation temperatures between 100 and 500 °C and the intensity has a maximum for hydrogenation at 250 °C.

Overexpression, crystallization and preliminary X-­ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

Science.gov (United States)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-01-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-­phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M) of 3.64 Å3 Da−1 and a solvent content of 66%. PMID:16511285

Hydrogen concentration control utilizing a hydrogen permeable membrane

International Nuclear Information System (INIS)

Keating, S.J. Jr.

1976-01-01

The concentration of hydrogen in a fluid mixture is controlled to a desired concentration by flowing the fluid through one chamber of a diffusion cell separated into two chambers by a hydrogen permeable membrane. A gradient of hydrogen partial pressure is maintained across the membrane to cause diffusion of hydrogen through the membrane to maintain the concentration of hydrogen in the fluid mixture at the predetermined level. The invention has particular utility for the purpose of injecting into and/or separating hydrogen from the reactor coolant of a nuclear reactor system

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Science.gov (United States)

Kawaguchi, Hideo; Katsuyama, Yohei; Danyao, Du; Kahar, Prihardi; Nakamura-Tsuruta, Sachiko; Teramura, Hiroshi; Wakai, Keiko; Yoshihara, Kumiko; Minami, Hiromichi; Ogino, Chiaki; Ohnishi, Yasuo; Kondo, Ahikiko

2017-07-01

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

Science.gov (United States)

Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

2016-01-01

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

The production of nitric oxide in EL4 lymphoma cells overexpressing growth hormone.

Science.gov (United States)

Arnold, Robyn E; Weigent, Douglas A

2003-01-01

Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by N(G)-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by L-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

Functional and gene expression analysis of hTERT overexpressed endothelial cells

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Haruna Takano

2008-09-01

Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma.

Science.gov (United States)

Lassus, Heini; Staff, Synnöve; Leminen, Arto; Isola, Jorma; Butzow, Ralf

2011-01-01

Aurora-A is a potential oncogene and therapeutic target in ovarian carcinoma. It is involved in mitotic events and overexpression leads to centrosome amplification and chromosomal instability. The objective of this study was to evaluate the clinical significance of Aurora-A and DNA ploidy in serous ovarian carcinoma. Serous ovarian carcinomas were analysed for Aurora-A protein by immunohistochemistry (n=592), Aurora-A copy number by CISH (n=169), Aurora-A mRNA by real-time PCR (n=158) and DNA ploidy by flowcytometry (n=440). Overexpression of Aurora-A was found in 27% of the tumors, cytoplasmic overexpression in 11% and nuclear in 17%. The cytoplasmic and nuclear overexpression were nearly mutually exclusive. Both cytoplasmic and nuclear overexpression were associated with shorter survival, high grade, high proliferation index and aberrant p53. Interestingly, only cytoplasmic expression was associated with aneuploidy and expression of phosphorylated Aurora-A. DNA ploidy was associated with poor patient outcome as well as aggressive clinicopathological parameters. In multivariate analysis, Aurora-A overexpression appeared as an independent prognostic factor for disease-free survival, together with grade, stage and ploidy. Aurora-A protein expression is strongly linked with poor patient outcome and aggressive disease characteristics, which makes Aurora-A a promising biomarker and a potential therapeutic target in ovarian carcinoma. Cytoplasmic and nuclear Aurora-A protein may have different functions. DNA aneuploidy is a strong predictor of poor prognosis in serous ovarian carcinoma. Copyright © 2010 Elsevier Inc. All rights reserved.

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

Science.gov (United States)

Wang, Jian-Feng; Xiong, Zhi-Qiang; Li, Shi-Yuan; Wang, Yong

2013-09-01

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

2008-08-01

tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

Elimination of Pathogen Escherichia coli O157:H7 in Ground Beef by a Newly Isolated Strain of Lactobacillus acidophilus during Storage at 5°C

Directory of Open Access Journals (Sweden)

Alireza Goodarzi

2016-06-01

Full Text Available Background and Objective: Constant use of limited number of lactic acid bacteria species in biopreservation can cause genetic degradation and or rising resistance against food pathogens or antimicrobial substances they produce. For this objective, a newly isolated strain of Lactobacillus acidophilus possessing high antimicrobial activity was evaluated as a candidate for use in biopreservation.Materials and Methods: Antibacterial activity was evaluated by agar disk diffusion method. Hydrogen peroxide amount was measured by Merckoquant Peroxide test strips. Microbiological analysis of the ground beef infected by Escherichia coli O157:H7 and treated by Lactobacillus acidophilus GH 201was done by plating of serial dilution in physiological saline on Tryptose agar.Results and Conclusion: The cells (109 CFU ml-1 of Lactobacillus acidophilus produced significant amount of antibacterial substances mainly hydrogen peroxide (28 and 30 μg ml-1 in sodium phosphate buffer (0.2 M, pH 6.5 and LAPTg at 5°C during submerged cultivation with no growth, respectively. Submerged co-cultivation of Escherichia coli O157:H7 with lactobacilli in LAPTg broth at 5°C reduced the total number of the pathogen more than 2 log for 5 days. In case of solid state cultivation on agar-based medium, the maximum inhibitory zones on Escherichia coli O157:H7 lawn around the disks soaked by different amounts of washed Lactobacillus acidophilus cells appear for one-day cold exposition. The size of inhibition zone depends on the concentration of lactic acid bacteria cells. The cell suspension intended for treatment must contain 108-9CFU ml-1 of lactic acid bacteria. Lactobacillus acidophilus reduced the initial amount (2×105 CFU ml-1 of Escherichia coli O157:H7 in ground beef up to 2 log for 5 days of solid-state co-cultivation. The application of Lactobacillus acidophilus bacteria expanded the shelf-life of ground beef due to inhibition of

Over-expression of HER-2 is associated with the stage in ...

African Journals Online (AJOL)

Background: The frequency of over-expression of human epidermal growth factor receptor-2 (HER-2) in bladder cancer is one of the highest among all human malignancies. This over-expression is thought to play a role in aberrant proliferation of cancer cells. Studies on HER-2 expression in bladder carcinoma have shown ...

Conjugation in Escherichia coli

Science.gov (United States)

Boyer, Herbert

1966-01-01

Boyer, Herbert (Yale University, New Haven, Conn.). Conjugation in Escherichia coli. J. Bacteriol. 91:1767–1772. 1966.—The sex factor of Escherichia coli K-12 was introduced into an E. coli B/r strain by circumventing the host-controlled modification and restriction incompatibilities known to exist between these closely related strains. The sexual properties of the constructed F+ B strain and its Hfr derivatives were examined. These studies showed that the E. coli strain B/r F+ and Hfr derivatives are similar to the E. coli strain K-12 F+ and Hfr derivatives. However, the site of sex factor integration was found to be dependent on the host genome. PMID:5327905

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins.

Science.gov (United States)

Yildirim, Deniz; Tükel, S Seyhan; Alagöz, Dilek

2014-01-01

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers.

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

Science.gov (United States)

2012-01-01

Background Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. Results Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. Conclusions In this work, we showed that Grx1 and

Long-term polarization of microglia upon alpha-synuclein overexpression in nonhuman primates

DEFF Research Database (Denmark)

Barkholt, Pernille; Sanchez-Guajardo, Vanesa Maria; Kirik, Denis

2012-01-01

We have previously shown that persistent ﰇ-sy- nuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) ﰇ-synuclein....

APRIL is overexpressed in cancer: link with tumor progression

International Nuclear Information System (INIS)

Moreaux, Jérôme; Veyrune, Jean-Luc; De Vos, John; Klein, Bernard

2009-01-01

BAFF and APRIL share two receptors – TACI and BCMA – and BAFF binds to a third receptor, BAFF-R. Increased expression of BAFF and APRIL is noted in hematological malignancies. BAFF and APRIL are essential for the survival of normal and malignant B lymphocytes, and altered expression of BAFF or APRIL or of their receptors (BCMA, TACI, or BAFF-R) have been reported in various B-cell malignancies including B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia. We compared the expression of BAFF, APRIL, TACI and BAFF-R gene expression in 40 human tumor types – brain, epithelial, lymphoid, germ cells – to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. We found significant overexpression of TACI in multiple myeloma and thyroid carcinoma and an association between TACI expression and prognosis in lymphoma. Furthermore, BAFF and APRIL are overexpressed in many cancers and we show that APRIL expression is associated with tumor progression. We also found overexpression of at least one proteoglycan with heparan sulfate chains (HS), which are coreceptors for APRIL and TACI, in tumors where APRIL is either overexpressed or is a prognostic factor. APRIL could induce survival or proliferation directly through HS proteoglycans. Taken together, these data suggest that APRIL is a potential prognostic factor for a large array of malignancies

Twinkle overexpression prevents cardiac rupture after myocardial infarction by alleviating impaired mitochondrial biogenesis.

Science.gov (United States)

Inoue, Takahiro; Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Matsuo, Yuka; Arai, Shinobu; Saku, Keita; Sunagawa, Kenji

2016-09-01

Cardiac rupture is a fatal complication after myocardial infarction (MI). However, the detailed mechanism underlying cardiac rupture after MI remains to be fully elucidated. In this study, we investigated the role of mitochondrial DNA (mtDNA) and mitochondria in the pathophysiology of cardiac rupture by analyzing Twinkle helicase overexpression mice (TW mice). Twinkle overexpression increased mtDNA copy number approximately twofold and ameliorated ischemic cardiomyopathy at day 28 after MI. Notably, Twinkle overexpression markedly prevented cardiac rupture and improved post-MI survival, accompanied by the suppression of MMP-2 and MMP-9 in the MI border area at day 5 after MI when cardiac rupture frequently occurs. Additionally, these cardioprotective effects of Twinkle overexpression were abolished in transgenic mice overexpressing mutant Twinkle with an in-frame duplication of amino acids 353-365, which resulted in no increases in mtDNA copy number. Furthermore, although apoptosis and oxidative stress were induced and mitochondria were damaged in the border area, these injuries were improved in TW mice. Further analysis revealed that mitochondrial biogenesis, including mtDNA copy number, transcription, and translation, was severely impaired in the border area at day 5 In contrast, Twinkle overexpression maintained mtDNA copy number and restored the impaired transcription and translation of mtDNA in the border area. These results demonstrated that Twinkle overexpression alleviated impaired mitochondrial biogenesis in the border area through maintained mtDNA copy number and thereby prevented cardiac rupture accompanied by the reduction of apoptosis and oxidative stress, and suppression of MMP activity. Copyright © 2016 the American Physiological Society.

A novel vector-based method for exclusive overexpression of star-form microRNAs.

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Bo Qu

Full Text Available The roles of microRNAs (miRNAs as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*, which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3'UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both in vitro and in vivo.

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

International Nuclear Information System (INIS)

Kiick, D.M.; Phillips, R.S.

1988-01-01

The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine

Hydrogen safety

International Nuclear Information System (INIS)

Frazier, W.R.

1991-01-01

The NASA experience with hydrogen began in the 1950s when the National Advisory Committee on Aeronautics (NACA) research on rocket fuels was inherited by the newly formed National Aeronautics and Space Administration (NASA). Initial emphasis on the use of hydrogen as a fuel for high-altitude probes, satellites, and aircraft limited the available data on hydrogen hazards to small quantities of hydrogen. NASA began to use hydrogen as the principal liquid propellant for launch vehicles and quickly determined the need for hydrogen safety documentation to support design and operational requirements. The resulting NASA approach to hydrogen safety requires a joint effort by design and safety engineering to address hydrogen hazards and develop procedures for safe operation of equipment and facilities. NASA also determined the need for rigorous training and certification programs for personnel involved with hydrogen use. NASA's current use of hydrogen is mainly for large heavy-lift vehicle propulsion, which necessitates storage of large quantities for fueling space shots and for testing. Future use will involve new applications such as thermal imaging

Escherichia coli pathotypes

Science.gov (United States)

Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

Hydrogen isotope effect through Pd in hydrogen transport pipe

International Nuclear Information System (INIS)

Tamaki, Masayoshi

1992-01-01

This investigation concerns hydrogen system with hydrogen transport pipes for transportation, purification, isotope separation and storage of hydrogen and its isotopes. A principle of the hydrogen transport pipe (heat pipe having hydrogen transport function) was proposed. It is comprised of the heat pipe and palladium alloy tubes as inlet, outlet, and the separation membrane of hydrogen. The operation was as follows: (1) gas was introduced into the heat pipe through the membrane in the evaporator; (2) the introduced gas was transported toward the condenser by the vapor flow; (3) the transported gas was swept and compressed to the end of the condenser by the vapor pressure; and (4) the compressed gas was exhausted from the heat pipe through the membrane in the condenser. The characteristics of the hydrogen transport pipe were examined for various working conditions. Basic performance concerning transportation, evacuation and compression was experimentally verified. Isotopic dihydrogen gases (H 2 and D 2 ) were used as feed gas for examining the intrinsic performance of the isotope separation by the hydrogen transport pipe. A simulated experiment for hydrogen isotope separation was carried out using a hydrogen-helium gas mixture. The hydrogen transport pipe has a potential for isotope separation and purification of hydrogen, deuterium and tritium in fusion reactor technology. (author)

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture ...

Pathogenic Escherichia coli and food handlers in luxury hotels in Nairobi, Kenya.

Science.gov (United States)

Onyango, Abel O; Kenya, Eucharia U; Mbithi, John J N; Ng'ayo, Musa O

2009-11-01

The epidemiology and virulence properties of pathogenic Escherichia coli among food handlers in tourist destination hotels in Kenya are largely uncharacterized. This cross-sectional study among consenting 885 food handlers working in nine luxurious tourist hotels in Nairobi, Kenya determined the epidemiology, virulence properties, antibiotics susceptibility profiles and conjugation abilities of pathogenic Escherichia coli. Pathogenic Escherichia coli was detected among 39 (4.4%) subjects, including 1.8% enteroaggregative Escherichia coli (EAEC) harboring aggR genes, 1.2% enterotoxigenic Escherichia coli (ETEC) expressing both LT and STp toxins, 1.1% enteropathogenic Escherichia coli (EPEC) and 0.2% Shiga-like Escherichia coli (EHEC) both harboring eaeA and stx2 genes respectively. All the pathotypes had increased surface hydrophobicity. Using multivariate analyses, food handlers with loose stools were more likely to be infected with pathogenic Escherichia coli. Majority 53.8% of the pathotypes were resistant to tetracycline with 40.2% being multi-drug resistant. About 85.7% pathotypes trans-conjugated with Escherichia coli K12 F(-) NA(r) LA. The carriage of multi-drug resistant, toxin expressing pathogenic Escherichia coli by this population is of public health concern because exposure to low doses can result in infection. Screening food handlers and implementing public awareness programs is recommended as an intervention to control transmission of enteric pathogens.

Overexpression of Insulin-like Growth Factor-1 Receptor Is Associated With Penile Cancer Progression.

Science.gov (United States)

Ball, Mark W; Bezerra, Stephania M; Chaux, Alcides; Faraj, Sheila F; Gonzalez-Roibon, Nilda; Munari, Enrico; Sharma, Rajni; Bivalacqua, Trinity J; Netto, George J; Burnett, Arthur L

2016-06-01

To evaluate insulin-like growth factor-1 receptor (IGF1R) expression in penile cancer and its association with oncologic outcomes. Tissue microarrays were constructed from 53 patients treated at our institution. Expression of IGF1R was evaluated using a Her2-like scoring system. Overexpression was defined as 1+ or greater membranous staining. Association of IGF1R expression with pathologic features was assessed with comparative statistics, and association with local recurrence, progression to nodal or distance metastases, or death was assessed with Kaplan-Meier survival analysis and Cox proportional hazard regression models. Overall, IGF1R overexpression was seen in 33 (62%) cases. With a median follow-up of 27.8 months, IGF1R overexpression was associated with inferior progression-free survival (PFS) (P  =  .003). In a multivariable model controlling for grade, T stage, perineural invasion, and lymphovascular invasion, IGF1R expression was independently associated with disease progression (hazard ratio 2.3, 95% confidence interval 1.1-5.1, P  =  .03. Comparing patients without IGF1R overexpression to those with overexpression, 5-year PFS was 94.1% vs 45.8%. IGF1R overexpression was associated with inferior PFS in penile cancer. Drugs that target IGF1R and downstream messengers may have a therapeutic benefit in patients that exhibit IGF1R overexpression. Copyright © 2016 Elsevier Inc. All rights reserved.

Production of soybean isoflavone genistein in non-legume plants via genetically modified secondary metabolism pathway.

Science.gov (United States)

Liu, Rongrong; Hu, Yuanlei; Li, Jialin; Lin, Zhongping

2007-01-01

Genetic modification of secondary metabolic pathways to produce desirable natural products is an attractive approach in plant biotechnology. In our study, we attempted to produce a typical soybean isoflavone genistein, a well-known health-promoting metabolite, in non-legume plants via genetic engineering. Both overexpression and antisense suppression strategies were used to manipulate the expression of several genes encoding key enzymes in the flavonoids/isoflavonoids pathway in transgenic tobacco, lettuce, and petunia. Introducing soybean isoflavone synthase (IFS) into these plants, which naturally do not produce isoflavonoids due to a lack of this leguminous enzyme, resulted in genistein biosynthesis in tobacco petals, petunia leaves and petals, and lettuce leaves. In tobacco, when flavanone 3-hydroxylase (F3H) expression was suppressed by its antisense gene while soybean IFS was overexpressed at the same time, genistein yield increased prominently. In addition, overexpression of phenylalanine ammonia-lyase (PAL) also led to an enhanced genistein production in tobacco petals and lettuce leaves in the presence of IFS than in the plants that overexpressed only IFS.

Chromatin architecture and gene expression in Escherichia coli

DEFF Research Database (Denmark)

Willenbrock, Hanni; Ussery, David

2004-01-01

Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

Overexpression of AIB1 in nasopharyngeal carcinomas correlates closely with advanced tumor stage.

Science.gov (United States)

Liu, Meng-Zhong; Xie, Dan; Mai, Shi-Juan; Tong, Zhu-Ting; Shao, Jian-Yong; Fu, Yong-Shui; Xia, Wen-Jie; Kung, Hsian-Fu; Guan, Xin-Yuan; Zeng, Yi-Xin

2008-05-01

AIB1, a candidate oncogene in breast cancer, is commonly amplified and overexpressed in several types of human cancers. In this study, expression and amplification of AIB1 in nasopharyngeal carcinoma (NPC) were studied by immunohistochemical analysis and fluorescence in situ hybridization using tissue microarrays, including 80 specimens of NPC and 20 specimens of nonneoplastic nasopharyngeal mucosa. In this NPC cohort, overexpression and amplification of AIB1 was detected in 36 (51%) of 71 and 3 (7%) of 46 NPCs, respectively. Overexpression of AIB1 was observed more frequently in NPCs in late T stages (T3/T4, 24/35 [69%]) than in earlier stages (T1/T2, 12/36 [33%]; P < .05). In addition, 18 (72%) of 25 NPCs with lymph node metastasis (N1-3) showed overexpression of AIB1; the frequency was significantly higher than that in NPCs without node metastasis (N0, 18/49 [39%]; P < .05). These findings suggest that overexpression of AIB1 in NPCs may be important in the acquisition of an invasive and/or metastatic phenotype.

Transcriptional regulation of Saccharomyces cerevisiaeCYS3 encoding cystathionine γ-lyase

Science.gov (United States)

Hiraishi, Hiroyuki; Miyake, Tsuyoshi

2008-01-01

In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine γ-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (−311 to −303) and a CGC motif (CGCCACAC; −193 to −186), which is one base shorter than the CCG motif, in the 5′-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; −217 to −210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs. PMID:18317767

Overexpression of BIRC6 Is a Predictor of Prognosis for Colorectal Cancer.

Directory of Open Access Journals (Sweden)

Tingting Hu

Full Text Available Inhibitors of apoptosis proteins (IAPs have been well investigated in human cancers, where they are frequently overexpressed and associated with poor prognosis. Here we explored the role of baculoviral IAP repeat containing 6 (BIRC6, a member of IAPs, in human colorectal cancer (CRC.We used Western blotting and immunohistochemistry to examine BIRC6 expression in 7 CRC cell lines and 126 CRC clinical samples. We determined the biological significance of BIRC6 in CRC cell lines by a lentivirus-mediated silencing method.We reported that BIRC6 was overexpressed in CRC cell lines and clinical CRC tissues. BIRC6 overexpression was correlated with tumor size and invasion depth of CRC. BIRC6 overexpression is associated with worse overall survival (OS (P = 0.001 and shorter disease-free survival (DFS (P = 0.010. BIRC6 knockdown inhibited cell proliferation, arrested cell cycle at S phase, downregulated cyclin A2, B1, D1 and E1 levels, and sensitized CRC cells to chemotherapy in vitro and in vivo.Taken together, these data suggests that BIRC6 overexpression is a predictor of poor prognosis in colorectal cancer and BIRC6 could be a potential target of CRC therapy.

Frequent Nek1 overexpression in human gliomas

Energy Technology Data Exchange (ETDEWEB)

Zhu, Jun [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Cai, Yu, E-mail: aihaozuqiu22@163.com [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Pin [Med-X Research Institute, Shanghai Jiao Tong University, Shanghai (China); Zhao, Weiguo [Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

2016-08-05

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Frequent Nek1 overexpression in human gliomas

International Nuclear Information System (INIS)

Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

2016-01-01

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Future hydrogen markets for large-scale hydrogen production systems

International Nuclear Information System (INIS)

Forsberg, Charles W.

2007-01-01

The cost of delivered hydrogen includes production, storage, and distribution. For equal production costs, large users (>10 6 m 3 /day) will favor high-volume centralized hydrogen production technologies to avoid collection costs for hydrogen from widely distributed sources. Potential hydrogen markets were examined to identify and characterize those markets that will favor large-scale hydrogen production technologies. The two high-volume centralized hydrogen production technologies are nuclear energy and fossil energy with carbon dioxide sequestration. The potential markets for these technologies are: (1) production of liquid fuels (gasoline, diesel and jet) including liquid fuels with no net greenhouse gas emissions and (2) peak electricity production. The development of high-volume centralized hydrogen production technologies requires an understanding of the markets to (1) define hydrogen production requirements (purity, pressure, volumes, need for co-product oxygen, etc.); (2) define and develop technologies to use the hydrogen, and (3) create the industrial partnerships to commercialize such technologies. (author)

Hydrogen embrittlement due to hydrogen-inclusion interactions

International Nuclear Information System (INIS)

Yu, H.Y.; Li, J.C.M.

1976-01-01

Plastic flow around inclusions creates elastic misfit which attracts hydrogen towards the regions of positive dilatation. Upon decohesion of the inclusion-matrix interface, the excess hydrogen escapes into the void and can produce sufficient pressure to cause void growth by plastic deformation. This mechanism of hydrogen embrittlement can be used to understand the increase of ductility with temperature, the decrease of ductility with hydrogen content, and the increase of ductility with the ultimate strength of the matrix. An examination of the effect of the shape of spheroid inclusion reveals that rods are more susceptible to hydrogen embrittlement than disks. The size of the inclusion is unimportant while the volume fraction of inclusions plays the usual role

Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

Directory of Open Access Journals (Sweden)

Kawe Martin

2009-01-01

Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.

Science.gov (United States)

Plaga, W; Vielhaber, G; Wallach, J; Knappe, J

2000-01-21

The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.

Generation of 2-Furfurylthiol by Carbon-Sulfur Lyase from the Baijiu Yeast Saccharomyces cerevisiae G20.

Science.gov (United States)

Zha, Musu; Sun, Baoguo; Yin, Sheng; Mehmood, Arshad; Cheng, Lei; Wang, Chengtao

2018-03-07

2-Furfurylthiol is the representative aroma compound of Chinese sesame-flavored baijiu. Previous studies demonstrated that baijiu yeasts could generate 2-furfurylthiol using furfural and l-cysteine as precursors and that the Saccharomyces cerevisiae genes STR3 and CYS3 are closely related to 2-furfurylthiol biosynthesis. To confirm the mechanism of the STR3- and CYS3-gene products on 2-furfurylthiol biosynthesis, their encoded proteins were purified, and we confirmed their activities as carbon-sulfur lyases. Str3p and Cys3p were able to cleave the cysteine-furfural conjugate to release 2-furfurylthiol. Moreover, the characterization of the enzymatic properties of the purified proteins shows good thermal stabilities and wide pH tolerances, which enable their strong potential for various applications. These data provide direct evidence that yeast Str3p and Cys3p release 2-furfurylthiol in vitro, which can be applied to improve baijiu flavor.

Hydrogen molecules and hydrogen-related defects in crystalline silicon

OpenAIRE

Fukata, N.; Sasak, S.; Murakami, K.; Ishioka, K.; Nakamura, K. G.; Kitajima, M.; Fujimura, S.; Kikuchi, J.; Haneda, H.

1997-01-01

We have found that hydrogen exists in molecular form in crystalline silicon treated with hydrogen atoms in the downstream of a hydrogen plasma. The vibrational Raman line of hydrogen molecules is observed at 4158cm-1 for silicon samples hydrogenated between 180 and 500 °C. The assignment of the Raman line is confirmed by its isotope shift to 2990cm-1 for silicon treated with deuterium atoms. The Raman intensity has a maximum for hydrogenation at 400 °C. The vibrational Raman line of the hydro...

Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases.

Science.gov (United States)

Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

2015-01-01

Upon recognition of microbe-associated molecular patterns (MAMPs) such as the bacterial flagellin (or the derived peptide flg22) by pattern-recognition receptors (PRRs) such as the FLAGELLIN SENSING2 (FLS2), plants activate the pattern-triggered immunity (PTI) response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2) is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs) possess two copies of the C-X8-C-X2-C (DUF26) motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here, we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6, and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1) was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6, and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity.

P53 overexpression and outcome of radiation therapy in head and neck cancers

International Nuclear Information System (INIS)

Kim, In Ah; Choi, Ihl Bhong; Kang, Ki Mun; Jang, Ji Young; Kim, Kyung Mi; Park, Kyung Shin; Kim, Young Shin; Kang, Chang Suk; Cho, Seung Ho; Kim, Hyung Tae

1999-01-01

Experimental studies have implicated the wild type p53 in cellular response to radiation. Whether altered p53 function can lead to changes in clinical radiocurability remains an area of ongoing study. This study was performed to investigate whether any correlation between change of p53 and outcome of curative radiation therapy in patients with head and neck cancers. Immunohistochemical analysis with a mouse monoclonal antibody (D0-7) specific for human p53 was used to detect to overexpression of protein in formalin fixed, paraffin-embedded tumor sample from 55 head and neck cancer patients treated with curative radiation therapy (median dose of 7020 cGy) from February 1988 to March 1996 at St. Mary's Hospital. Overexpression of p53 was correlated with locoregional control and survival using Kaplan-Meier method. A Cox regression multivariate analysis was performed that included all clinical variables and status of p53 expression. Thirty-seven (67.2%) patients showed overexpression of p53 by immunohistochemical staining in their tumor. One hundred percent of oral cavity, 76% of laryngeal, 66.7% of oropharyngeal, 66.7% of hypopharyngeal cancer showed p53 overexpression (p=0.05). The status of p53 had significant relationship with stage of disease (p=0.03) and history of smoking (p=0.001). The overexpression of p53 was not predictive of response rate to radiation therapy. The locoregional control was not significantly affected by p53 status. Overexpression of p53 didn't have any prognostic implication for disease free survival and overall survival. Primary site and stage of disease were significant prognostic factors for survival. The p53 overexpression as detected by immunohistochemical staining had significant correlation with stage, primary site of disease and smoking habit of patients. The p53 overexpression didn't have any predictive value for outcome of curative radiation therapy in a group of head and neck cancers

P53 overexpression and outcome of radiation therapy in head and neck cancers

Energy Technology Data Exchange (ETDEWEB)

Kim, In Ah; Choi, Ihl Bhong; Kang, Ki Mun; Jang, Ji Young; Kim, Kyung Mi; Park, Kyung Shin; Kim, Young Shin; Kang, Chang Suk; Cho, Seung Ho; Kim, Hyung Tae [College of Medicine, The Catholic Univ., Seoul (Korea, Republic of)

1999-03-01

Experimental studies have implicated the wild type p53 in cellular response to radiation. Whether altered p53 function can lead to changes in clinical radiocurability remains an area of ongoing study. This study was performed to investigate whether any correlation between change of p53 and outcome of curative radiation therapy in patients with head and neck cancers. Immunohistochemical analysis with a mouse monoclonal antibody (D0-7) specific for human p53 was used to detect to overexpression of protein in formalin fixed, paraffin-embedded tumor sample from 55 head and neck cancer patients treated with curative radiation therapy (median dose of 7020 cGy) from February 1988 to March 1996 at St. Mary's Hospital. Overexpression of p53 was correlated with locoregional control and survival using Kaplan-Meier method. A Cox regression multivariate analysis was performed that included all clinical variables and status of p53 expression. Thirty-seven (67.2%) patients showed overexpression of p53 by immunohistochemical staining in their tumor. One hundred percent of oral cavity, 76% of laryngeal, 66.7% of oropharyngeal, 66.7% of hypopharyngeal cancer showed p53 overexpression (p=0.05). The status of p53 had significant relationship with stage of disease (p=0.03) and history of smoking (p=0.001). The overexpression of p53 was not predictive of response rate to radiation therapy. The locoregional control was not significantly affected by p53 status. Overexpression of p53 didn't have any prognostic implication for disease free survival and overall survival. Primary site and stage of disease were significant prognostic factors for survival. The p53 overexpression as detected by immunohistochemical staining had significant correlation with stage, primary site of disease and smoking habit of patients. The p53 overexpression didn't have any predictive value for outcome of curative radiation therapy in a group of head and neck cancers.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

Energy Technology Data Exchange (ETDEWEB)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

2006-02-01

Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B{sub 6} (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.64 Å{sup 3} Da{sup −1} and a solvent content of 66%.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-01-01

Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B 6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M ) of 3.64 Å 3 Da −1 and a solvent content of 66%

Why hydrogen; Pourquoi l'hydrogene?

Energy Technology Data Exchange (ETDEWEB)

NONE

2004-02-01

The energy consumption increase and the associated environmental risks, led to develop new energy sources. The authors present the potentialities of the hydrogen in this context of energy supply safety. They detail the today market and the perspectives, the energy sources for the hydrogen production (fossils, nuclear and renewable), the hydrogen transport, storage, distribution and conversion, the application domains, the associated risks. (A.L.B.)

Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency

Directory of Open Access Journals (Sweden)

Mónica Fernández-Cancio

2018-04-01

Full Text Available The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Science.gov (United States)

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.