scholarly journals Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production inHevea brasiliensis

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Wei Fang ◽  
Luo Shi Qiao ◽  
Wu Ming ◽  
Qiu Jian ◽  
Yang Wen Feng ◽  
...  
Keyword(s):  
Specific Activity ◽  
Rubber Tree ◽  
Natural Rubber Latex ◽  
Cloning And Expression ◽  
E Coli ◽  
Glutamylcysteine Synthetase ◽  
Commercial Source ◽  
Rate Limiting

Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme,γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation betweenγ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of oneγ-ECS from rubber tree latex (Hbγ-ECS1). According to qPCR analysis, the expression levels ofHbγ-ECS1were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression ofHbγECS1increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow.HbγECS1was expressed inE. coliRosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827 U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role ofHbγ-ECSin thiols biosynthesis, which is influenced by tapping.

Effects of nonsulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism

1999 ◽  
Vol 277 (1) ◽  
pp. E144-E153 ◽  
Author(s):  
Deborah L. Bella ◽  
Christine Hahn ◽  
Martha H. Stipanuk
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Mrna Level ◽  
Basal Diet ◽  
Cysteine Dioxygenase ◽  
Sulfur Amino Acids ◽  
Subunit Mrna ◽  
Cysteine Metabolism ◽  
Glutamylcysteine Synthetase

To determine the role of nonsulfur vs. sulfur amino acids in regulation of cysteine metabolism, rats were fed a basal diet or diets supplemented with a mixture of nonsulfur amino acids (AA), sulfur amino acids (SAA), or both for 3 wk. Hepatic cysteine-sulfinate decarboxylase (CSDC), cysteine dioxygenase (CDO), and γ-glutamylcysteine synthetase (GCS) activity, concentration, and mRNA abundance were measured. Supplementation with AA alone had no effect on any of these measures. Supplementation of the basal diet with SAA, with or without AA, resulted in a higher CDO concentration (32–45 times basal), a lower CSDC mRNA level (49–64% of basal), and a lower GCS-heavy subunit mRNA level (70–76%). The presence of excess SAA and AA together resulted in an additional type of regulation: a lower specific activity of all three enzymes was observed in rats fed diets with an excess of AA and SAA. Both SAA and AA played a role in regulation of these three enzymes of cysteine metabolism, but SAA had the dominant effects, and effects of AA were not observed in the absence of SAA.

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

scholarly journals Plasmid DNA Supercoiling and Survival in Long-Term Cultures of Escherichia coli: Role of NaCl

2003 ◽  
Vol 185 (17) ◽  
pp. 5324-5327 ◽  
Author(s):  
Annie Conter
Keyword(s):  
Escherichia Coli ◽  
Dna Supercoiling ◽  
Luria Bertani ◽  
Plasmid Pbr322 ◽  
E Coli ◽  
Topological State ◽  
Negative Supercoiling ◽  
The Relationship

ABSTRACT The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.

Physiological and pharmacologic effects on TH activity in rabbit and cat carotid body

1981 ◽  
Vol 240 (1) ◽  
pp. R38-R43 ◽  
Author(s):  
C. Gonzalez ◽  
Y. Kwok ◽  
J. Gibb ◽  
S. Fidone
Keyword(s):  
Carotid Body ◽  
Species Differences ◽  
Adrenal Glands ◽  
Catecholamine Biosynthesis ◽  
Carotid Bodies ◽  
Cervical Ganglion ◽  
Cervical Ganglia ◽  
Rate Limiting

The carotid bodies, along with the superior cervical ganglia and the adrenal glands, were removed from rabbits and cats and the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, was assayed by the method of Nagatsu (Anal. Biochem. 9: 122-126, 1964). The activities of the enzyme, in nmols tyrosine hydroxylated x h-1 x mg tissue-1, were: carotid body, rabbit 1.29, cat 0.84; superior cervical ganglion, rabbit 8.66, cat 4.97; adrenal gland, rabbit 0.95, cat 2.25. With respect to the carotid body, each of the following experimental procedures resulted in a long-term increase in TH activity in the rabbit but not in the cat: 1) severe hypoxia (5% O2 in N2 for 1 h, assay of TH 48 h later); 2) chronic transection of the carotid sinus nerve (assay of TH at 12-15 days); or 3) administration of reserpine (10 mg/kg at 0 and 24 h, assay of TH at 48 h). These observations are compared with our previous findings for rat carotid body and are discussed in relation to the role of catecholamines in chemoreception, and, in particular, to the reported differences in dopamine action in the carotid bodies of these different species. Our results also suggest species differences with respect to the participation of the sympathoadrenal system in response to reserpine and hypoxic stress.

Characterization and Functional Expression of Xylose Isomerase from Thermus thermophilus

2013 ◽  
Vol 641-642 ◽  
pp. 919-922
Author(s):  
An Gen Lu ◽  
Ze Xi Yang ◽  
Fei Wang ◽  
Lang Xu ◽  
Wen Ying Deng ◽  
...  
Keyword(s):  
Fossil Fuels ◽  
Specific Activity ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Functional Expression ◽  
Future Application ◽  
Gene Encoding ◽  
E Coli ◽  
Rate Limiting ◽  
Pentose Sugars

Ethanol produced from hexose and pentose sugars hydrolysated by lignocellulose is an environment-friendly alternative to fossil fuels. Xylose isomerase is the major rate-limiting enzyme in the ethanol synthesis biologically pathway of xylose fermentation. In present study, xylA gene encoding xylose isomerase was cloned from Thermus thermophilus and overexpressed in E. coli BL21. Purified recombinant enzyme was used to study the enzymatic characterization. Specific activity of recombinant PDOR was 19.6 U/mg. Optimal temperature and pH were 80 °C, 8.0, respectively. Km and Vmax values were 15.9 mM, 22.8 U/mg. This research may form a basis for the future application of xylose isomerase.

scholarly journals A long-term epigenetic memory switch controls bacterial virulence bimodality

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Irine Ronin ◽  
Naama Katsowich ◽  
Ilan Rosenshine ◽  
Nathalie Q Balaban
Keyword(s):  
Mathematical Modeling ◽  
Virulence Genes ◽  
Phenotypic Variability ◽  
Bacterial Virulence ◽  
Environmental Cues ◽  
Human Pathogen ◽  
Epigenetic Memory ◽  
E Coli

When pathogens enter the host, sensing of environmental cues activates the expression of virulence genes. Opposite transition of pathogens from activating to non-activating conditions is poorly understood. Interestingly, variability in the expression of virulence genes upon infection enhances colonization. In order to systematically detect the role of phenotypic variability in enteropathogenic E. coli (EPEC), an important human pathogen, both in virulence activating and non-activating conditions, we employed the ScanLag methodology. The analysis revealed a bimodal growth rate. Mathematical modeling combined with experimental analysis showed that this bimodality is mediated by a hysteretic memory-switch that results in the stable co-existence of non-virulent and hyper-virulent subpopulations, even after many generations of growth in non-activating conditions. We identified the per operon as the key component of the hysteretic switch. This unique hysteretic memory switch may result in persistent infection and enhanced host-to-host spreading.

scholarly journals Changes in ATP Sulfurylase activity in response to altered cyanobacteria growth conditions

2020 ◽  
Author(s):  
Lucia Gastoldi ◽  
Lewis M. Ward ◽  
Mayuko Nakagawa ◽  
Mario Giordano ◽  
Shawn E. McGlynn
Keyword(s):  
Growth Rate ◽  
Cell Growth ◽  
Redox Regulation ◽  
Specific Activity ◽  
Growth Conditions ◽  
Cell Physiology ◽  
Atp Sulfurylase ◽  
Environmental Media ◽  
Rate Limiting

Here we investigated variations in cell growth and ATP sulfurylase activity when two cyanobacterial strains – Synechocystis sp. PCC6803 and Synechococcus sp. WH7803 – were grown comparatively between conventional media and media with low ammonium, low sulfate and a controlled high CO2/low O2 atmosphere, which might resemble some Precambrian environments. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS specific activity. This decrease in activity appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and thorughout history.

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12

1982 ◽  
Vol 152 (3) ◽  
pp. 1138-1146
Author(s):  
L J Lee ◽  
J B Hansen ◽  
E K Jagusztyn-Krynicka ◽  
B M Chassy
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Protein Product ◽  
Coli Strain ◽  
Cloning And Expression ◽  
E Coli ◽  
Lactose Metabolism ◽  
Gene Coding ◽  
K 12

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

Cloning and expression of DL-2-haloacid dehalogenase gene from Burkholderia cepacia

2000 ◽  
Vol 42 (7-8) ◽  
pp. 261-268 ◽  
Author(s):  
Y. Ohkouchi ◽  
H. Koshikawa ◽  
Y. Terashima
Keyword(s):  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Wild Strain ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Vector Systems ◽  
Lac Promoter ◽  
Acetic Acids

Burkholderia cepacia strain KY, which can utilize a herbicide 2,4-D as a sole carbon and energy source, catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids. We have cloned the gene encoding DL-2-haloacid dehalogenase, and obtained a recombinant plasmid (pUCDEXL) containing approximately 4.5 kbp insert. In both of B. cepacia strain KY and this clone E. coli JM109/pUCDEXL, DL-2-haloacid dehalogenase was induced significantly with monohalogenated acetic acids, such as chloroacetate, bromoacetate and iodoacetate. This dehalogenase was also overexpressed in E. coli using three different promoters. In pET vector systems with T7 lac promoter, a large amount of dehalogenase was selectively expressed, but some parts of the protein were accumulated in the form of inclusion bodies. This problem was overcome to carry on growth and induction at 22°C, and at the same time, the maximum specific activity of dehalogenase was reached at 12.6 U/mg, 500-fold higher activity than in wild strain, B. cepacia strain KY grown with 2,4-D.

A New Functional Role of 5,6,7,8-tetrahydrobiopterin (BH4) as an Inhibitor of γ-glutamylcysteine Synthetase (γ-GCS) mRNA Expression

Pteridines ◽  
2002 ◽  
Vol 13 (3) ◽  
pp. 83-88
Author(s):  
Shuji Kojima ◽  
Risa Kurozumi ◽  
Maiko Tuchiya
Keyword(s):  
Hydrogen Peroxide ◽  
Mrna Expression ◽  
Functional Role ◽  
De Novo ◽  
Dependent Manner ◽  
Glutathione Synthesis ◽  
Glutamylcysteine Synthetase ◽  
Rate Limiting ◽  
Dose Dependent

Abstract The effect of the intracellular 5,6,7,8-tetrahydrobiopterin (BH4) on the increased γ-glutamylcysteine synthetase (γ-GCS) mRNA expression induced by nitric oxide (NO) was investigated in RAW264.7 cells. Low doses of nitroprusside (SNP), ranging from 0.1 mM to 0.5 mM, significantly increased the intracellular glutathione levels along with the expression of mRNA for γ-GCS, a rate limiting enzyme of de novo glutathione synthesis. The increased expression was not abolished by exogenously added BH4 itself, but by sepiapterin, a precursor of BH4 synthesis, in a dose-dependent manner. The blockage by sepiapterin was ineffective in the presence of N-acetyl serotonin (NAS), an inhibitor of sepiapterin reductase. The increased γ-GCS mRNA expression was also inhibited by catalase, a scavenger of hydrogen peroxide. Evidence was further provided that BH4 effectively scavenged hydrogen peroxide. These results suggest that intracellular BH4 may play a role as an inhibitor of glutathione synthesis induced by NO via scavenging hydrogen peroxide, a mediator of the gene expression for the de novo glutathione synthesis pathway.

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