Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

A MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis.

Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium (Cd) stress. However, the underlying mechanisms and signaling pathways involved are poorly understood. In this study, we showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance. Loss-of-function of MNB1 (mannose-binding-lectin 1) led to decreased Cd accumulation and tolerance, whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance. Consistently, expression of the genes involved in the GSH-dependent phytochelatin (PC) synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress, which was positively correlated with Cd-activated PC synthesis. Moreover, we found that mannose is able to bind to the GNA-related domain of MNB1, and that mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance in Arabidopsis. Further analysis showed that MYB4 directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 and thus Cd tolerance via the GSH-dependent PC synthesis pathway. Consistent with these findings, overexpression of MAN3 rescued the Cd-sensitive phenotype of the myb4 mutant but not the mnb1 mutant, whereas overexpression of MNB1 rescued the Cd-sensitive phenotype of the myb4 mutant. Taken together, our results provide compelling evidence that a MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.

Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

Characterization of glutamate-cysteine ligase and glutathione synthetase from the δ-proteobacterium Myxococcus xanthus.

Glutathione (GSH) is synthesized in two ATP-dependent reactions by glutamate-cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram-negative bacterium belonging to δ-proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+ , but not by Mg2+ , and stabilized in the presence of 5 mM Mn2+ or Mg2+ . Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki  = 2.1 µM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+ . MxGs was inhibited by GSSG at Ki  = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.

Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation.

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene.

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to "GS-like effectors". Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function.

Spatial locations of certain enzymes and transporters within preinvasive ductal epithelial cells predict human breast cancer recurrences.

Some patients treated for ductal carcinoma in situ (DCIS) of the breast will experience cancer recurrences, whereas other patients will not. Unfortunately, current techniques cannot identify which preinvasive lesions will lead to recurrent cancer. Because the mechanism of cancer recurrence is unknown, it is difficult to design a test that detects its activity. We propose that certain pentose phosphate pathway enzymes, glutathione synthesis enzymes, and RhoA cluster at the epithelial cell periphery before cancer recurrences. Enzyme clustering enhances metabolic flux. Using fluorescence microscopy, we show that phosphophorylated glucose transporter type-1, transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and RhoA accumulate as a peripheral layer near the epithelial cell surface in surgical biopsies of women who will suffer recurrences, but not in samples from women who will not experience recurrences as judged using 2×2 contingency tables. Machine-learning studies of phospho-glucose transporter type 1-labeled tissue sections of patients with DCIS demonstrated strong cross-validation and holdout performance. A machine study of individual cribriform, papillary, micropapillary, and comedo forms of DCIS demonstrated 97% precision and 95% recall in the detection of samples from women who will not experience a recurrence and 90% precision and 94% recall in the detection of lesions that will become recurrent. A holdout study of these patients showed 73% true negatives, 18% true positives, 4% false positives, and 4% false negatives at a 50% threshold. This work suggests mechanistic features of cancer recurrences that may contribute to a new clinical test distinguishing high from low-recurrence risk in patients with DCIS.

Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes.

2-Phenylethanol (2-PE) is an important flavor compound but also impairs cell growth severely, which in turn blocks its bioproduction. However, the molecular mechanism of 2-PE tolerance is unclear. In this study, a superb 2-PE stress-tolerant and producing yeast, Candida glycerinogenes, was selected to uncover the underlying mechanism of 2-PE tolerance. We discovered that Hap5 is an essential regulator to 2-PE resistance, and its induction by 2-PE stress occurs at the post-transcriptional level, rather than at the transcriptional level. Under 2-PE stress, Hap5 is activated and imported into the nucleus rapidly. Then, the nuclear Hap5 binds to the glutathione synthetase (gsh2) promoter via CCAAT box, to induce the expression of gsh2 gene. The increased gsh2 expression contributes to enhanced cellular glutathione content, and consequently alleviates ROS accumulation, lipid peroxidation, and cell membrane damage caused by 2-PE toxicity. Specifically, increasing the expression of gsh2 is effective in improving not just 2-PE tolerance (33.7% higher biomass under 29 mM 2-PE), but also 2-PE production (16.2% higher). This study extends our knowledge of 2-PE tolerance mechanism and also provides a promising strategy to improve 2-PE production.

Inhibition of connexin 43 attenuates oxidative stress and apoptosis in human umbilical vein endothelial cells.

BACKGROUND: Previous studies demonstrated an important role for connexin 43 (Cx43) in the regulation of apoptosis by influencing mitochondrial functions. This study aimed to investigate the relationship between Cx43 and lipopolysaccharide (LPS)-induced oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS: Western blot was performed to determine mitochondrial Cx43 (MtCx43) protein level and phosphorylation (p-MtCx43). Gap19, a selective Cx43 inhibitor, was used to examine the effects of Cx43 on LPS-induced oxidative stress and apoptosis in HUVECs. Expression of regulatory genes associated with oxidative stress was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Apoptosis was assessed by flow cytometry. RESULTS: LPS stimulation resulted in increased levels of MtCx43 and p-MtCx43. Interestingly, Gap19 antagonized the upregulation of glutathione S-transferase Zeta 1 (GSTZ1) and cytochrome b alpha beta (CYBB), and the downregulation of antioxidant 1 (ATOX1), glutathione synthetase (GSS) and heme oxygenase 1 (HMOX1) induced by LPS or Cx43 overexpression. Moreover, the increased production of reactive oxygen species (ROS) and apoptosis elicited by LPS or Cx43 overexpression were reduced following treatment with Gap19. CONCLUSIONS: Selective inhibition of Cx43 hemichannels protects HUVECs from LPS-induced apoptosis and this may be via a reduction in oxidative stress production.

Ascorbic acid ameliorates dysregulated folliculogenesis induced by mono-(2-ethylhexyl)phthalate in neonatal mouse ovaries via reducing ovarian oxidative stress.

Phthalates, including di-(2-ethylhexyl)phthalate (DEHP), are common industrial chemicals in the environment. Recent evidence indicates that DEHP and its active metabolite mono-(2-ethylhexyl)phthalate (MEHP) negatively modulate reproductive functions and induce reactive oxygen species. Ascorbic acid (AA) is a dietary requirement for primates, and it acts as a potent free radical scavenger to protect tissues against oxidative stress. In this study, to investigate the toxic effects of MEHP on the follicle development and the beneficial role of AA, neonatal mouse ovaries were treated with different concentrations of MEHP with or without AA for 6 days. Then, the follicle constitution and oxidative status were compared in different groups. Results showed MEHP accelerated primordial follicle recruitment by increasing the percentage of primary and secondary follicles and decreasing the percentage of primordial follicles in the ovaries. Moreover, MEHP-induced ovarian oxidative stress by significantly increasing malondialdehyde (MDA) concentration and the expression of GSS and SOD1. When ovaries were co-administrated with MEHP and AA, follicle constitution was normalized, and the oxidative status was significantly decreased. These results suggested that AA ameliorated MEHP-induced ovarian oxidative stress and follicular dysregulation, which attested the clinical significance of AA for ovary protection in the case of MEHP exposure.

Glutathione synthetase deficiency: a novel mutation with femur agenesis.

Introduction: Glutathione synthetase (GSS) deficiency is an autosomal recessive disorder (frequency < 1/1,000,000) with different varyingly severe clinical manifestations that include metabolic acidosis, hemolytic anemia, hyperbilirubinemia, neurological disorders and sepsis. Case report: This infant was small for gestational age, had hemolytic anemia, metabolic acidosis, bilateral subependymal pseudocysts and increased echogenicity of the basal ganglia. GSS deficiency was confirmed by genetic analysis. The patient also had unilateral right femur agenesis. Conclusion: By using next generation sequencing analysis, we identified a novel homozygous variant c.800G > A, p.Arg267Gln in the GSS gene of this patient. Femur agenesis had not previously been associated with GSS.

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels.IMPORTANCEPseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Cysteine homeostasis under inhibition of protein synthesis in Escherichia coli cells.

Increased intracellular cysteine poses a potential danger to cells due to the high ability of cysteine to reduce free iron and promote the Fenton reaction. Here, we studied ways to maintain cysteine homeostasis in E. coli cells while inhibiting protein synthesis with valine or chloramphenicol. When growing wild-type bacteria on minimal medium with sulfate, an excess of cysteine resulting from the inhibition of protein synthesis is mainly incorporated into glutathione (up to 90%), which, therefore, can be considered as cysteine buffer. The share of hydrogen sulfide, which is the product of cysteine degradation by cysteine synthase B (CysM), does not exceed 1-3%, the rest falls on free cysteine, exported from cells. As a result, intracellular free cysteine is maintained at a low level (about 0.1 mM). The lack of glutathione in the gshA mutant increases H2S production and excretion of cysteine and leads to a threefold increase in the level of intracellular cysteine in response to valine and chloramphenicol. The relA mutants, exposed to valine, produce more H2S, dramatically accelerate the export of glutathione and accumulate more cysteine in the cytoplasm than their parent, which indicates that the regulatory nucleotide (p)ppGpp is involved in maintaining cysteine homeostasis. Disruption of cysteine homeostasis in gshA and relA mutants increases their sensitivity to peroxide stress.

Sustainable production of glutathione from lignocellulose-derived sugars using engineered Saccharomyces cerevisiae.

Glutathione has diverse physiological functions, and therefore, the demand for it has increased recently. Currently, industrial mass production of glutathione is performed from D-glucose via fermentation by the budding yeast Saccharomyces cerevisiae. However, use of D-glucose often competes with demands for various other industries, leading to high production costs. To affordably produce glutathione, we aimed to produce high amounts of glutathione from D-glucose and D-xylose, which are the main constituents of lignocellulosic biomass pre-treated with acids. Genetically engineered S. cerevisiae strains that can produce high amounts of glutathione and assimilate D-xylose were constructed and cultured in media containing D-xylose. Among these recombinant strains, a S. cerevisiae GCI (XR/XDH/XK) strain over-expressing γ-glutamylcysteine synthetase, glutathione synthetase, D-xylose reductase, xylitol dehydrogenase, and xylulokinase genes successfully consumed D-xylose in the medium and produced the highest amount of glutathione. When strains were grown in media containing D-glucose and D-xylose, the GCI (XR/XDH/XK) strain showed 4.6-fold higher volumetric glutathione production (mg/L-broth), 2.2-fold higher glutathione content (%), and 2.1-fold higher cell growth (g-cell/L-broth) than the vector control strain of YPH499 (Vector). Furthermore, when recombinant S. cerevisiae strains were grown in medium containing fermentation inhibitory materials, the GCI (XR/XDH/XK) strain produced 5.8- and higher volumetric glutathione, 2.6-fold higher intracellular glutathione, and 2.9-fold higher cell growth than the vector control YPH499 (Vector) strain. The gradual sugar consumption by recombinant S. cerevisiae strains in medium containing D-glucose and D-xylose leads to high yields of glutathione. These results indicate the potential for glutathione production from lignocellulosic materials.

Cloning and Analysis of Genes Controlling Antibacterial Activities of Burkholderia pyrrocinia Strain Lyc2.

The Burkholderia pyrrocinia Lyc2 strain isolated from healthy plant rhizosphere showed significant antimicrobial activities against a variety of plant pathogens. In this study, a random mutation library was constructed using an EZ-Tn5 transposome kit and Erwinia amylovora was used as an indicator to screen for mutants with defective antibacterial activity. The transposon gene was verified in the chromosome of the Lyc2 strain using polymerase chain reaction (PCR). The gene that was disrupted by transposon was amplified by rescue cloning for functional and bioinformatics analyses. Antibacterial analysis indicated that the mutant Lyc2-MT2918 was defective in antibacterial activity. Sequence alignment of the mutant suggested that the disrupted gene Glu-2918 was homologous to the glutathione (GSH) synthase gene Bamb-2918 of strain B. ambifaria AMMD. Genetic functional analysis and complementary assay of the disrupted gene, which was predicted to encode GSH synthase, indicated the essential role of the Glu-2918 gene in the antibacterial activity of strain Lyc2.

Comparative Transcriptome Analysis of the Molecular Mechanism of the Hairy Roots of Brassica campestris L. in Response to Cadmium Stress.

Brassica campestris L., a hyperaccumulator of cadmium (Cd), is considered a candidate plant for efficient phytoremediation. The hairy roots of Brassica campestris L are chosen here as a model plant system to investigate the response mechanism of Brassica campestris L. to Cd stress. High-throughput sequencing technology is used to identify genes related to Cd tolerance. A total of 2394 differentially expressed genes (DEGs) are identified by RNA-Seq analysis, among which 1564 genes are up-regulated, and 830 genes are down-regulated. Data from the gene ontology (GO) analysis indicate that DEGs are mainly involved in metabolic processes. Glutathione metabolism, in which glutathione synthetase and glutathione S-transferase are closely related to Cd stress, is identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. A Western blot shows that glutathione synthetase and glutathione S-transferase are involved in Cd tolerance. These results provide a preliminary understanding of the Cd tolerance mechanism of Brassica campestris L. and are, hence, of particular importance to the future development of an efficient phytoremediation process based on hairy root cultures, genetic modification, and the subsequent regeneration of the whole plant.

Association of mRNA Levels of IL6, MMP-8, GSS in Saliva and Pyelonephritis in Children.

Nowadays, saliva is a subject of growing scientific interest because of its definite advantages as diagnostic medium. The aim of our study was to investigate the diagnostic potential and reliability of messenger RNAs (mRNAs) of selected genes-interleukin-6 (IL-6), matrix metalloproteinase-8 (MMP-8) and glutathione synthetase (GSS)-as salivary markers in children with diagnosed pyelonephritis and to correlate their levels with typical urine para-clinical indicators of the disease. Analysis of the mRNA levels for IL-6, MMP-8 and GSS in 28 children hospitalized with the diagnosis of pyelonephritis was conducted applying the method of quantitative reverse transcription polymerase chain reaction (RT-qPCR). In the study group (n = 28), IL-6 mRNA levels demonstrated 64-fold increase (p < 0.001). MMP-8 and GSS mRNA levels were increased in 12 samples in patients with pyelonephritis 3.27 (p < 0.01) and 1.94 (p < 0.001) times, respectively. We found a strong and significant correlation (p < 0.001) between the investigated mRNA for IL-6 and MMP-8, IL-6 and GSS, MMP-8 and GSS. Moderate degree of correlation was established between IL-6 and the typical para-clinical indicator of leucocytes (0.43, p < 0.05) and between GSS and leucocytes (0.54, p < 0.01). Salivary IL-6, MMP-8 and GSS mRNA levels in combination with urine test analysis could be useful diagnostic tool for the very distributed disorder of pyelonephritis in childhood.

Jasmonate Regulates Plant Responses to Postsubmergence Reoxygenation through Transcriptional Activation of Antioxidant Synthesis.

Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis.

Effects of cadmium exposure on expression of glutathione synthetase system genes in Acidithiobacillus ferrooxidans.

The glutathione synthetase system (GSS) is an important pathway of glutathione synthesis and plays a key role in heavy metal resistance. In this work, the response of Acidithiobacillus ferrooxidans to extracellular Cd2+ was investigated, and the interplay between Cd2+ resistance and the expression of GSS related-genes was analyzed by reverse-transcription quantitative PCR (RT-PCR). During growth in the presence of 5, 15 and 30 mM Cd2+, the transcript levels of eight GSS pathway genes were affected between 0.81- and 7.12-fold. Increased transcription was also reflected in increased enzyme activities: with those of glutathione reductase (GR) increased by 1.10-, 2.26- and 1.54-fold in the presence of 5, 15 and 30 mM Cd2+, respectively. In contrast, the activities of catalase (CAT) and superoxide dismutase (SOD) were decreased in the presence of Cd2+. At the metabolite level, intracellular methane dicarboxylic aldehyde (MDA) content was increased 1.97-, 3.31- and 1.92-fold in the presence of 5, 15 and 30 mM Cd2+, respectively. These results suggest that Cd2+ directly inhibits the activities of CAT and SOD, breaks the redox balance of the cells, which leads to the activation of the other antioxidant pathway of GSS. Resistance of A. ferrooxidans to Cd2+ may involve modulation of expression levels of glutathione S-transferase (GST), GR, and glutathione synthetase, which may protect against oxidative damage.