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.

Clickable Glutathione-Based Identification of Cysteine Glutathionylation.

Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells.

Gas Chromatography Mass Spectrometry Aided Diagnosis of Glutathione Synthetase Deficiency.

Glutathione synthetase (GSS) deficiency is a rare disorder, occurring with a frequency of less than 1 in 100,000 individuals worldwide. The clinical presentation may vary from mild to severe, and manifestations include hemolytic anemia, hyperbilirubinemia, metabolic acidosis, neurological problems, and sepsis. Herein, we present a case of a newborn boy with the most severe phenotype of GSS deficiency, diagnosed based on clinical features and increased urinary 5-oxoproline levels determined via gas chromatography mass spectrometry (GCMS) testing.

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.

Susceptibility of Oocytes from Gilts and Sows to Beauvericin and Deoxynivalenol and Its Relationship with Oxidative Stress.

Beauvericin (BEA) and deoxynivalenol are toxins produced by Fusarium species that can contaminate food and feed. The aim of this study was to assess the effects of these mycotoxins on the maturation of oocytes from gilts and sows. Furthermore, the antioxidant profiles in the oocytes' environment were assessed. Cumulus-oocyte-complexes (COCs) from gilts and sows were exposed to beauvericin (BEA) or deoxynivalenol (DON) and matured in vitro. As an extra control, these COCs were also exposed to reactive oxygen species (ROS). The maturation was mostly impaired when oocytes from gilts were exposed to 0.02 µmol/L DON. Oocytes from sows were able to mature even in the presence of 5 µmol/L BEA. However, the maturation rate of gilt oocytes was already impaired by 0.5 µmol/L BEA. It was observed that superoxide dismutase (SOD) and glutathione (GSH) levels in the follicular fluid (FF) of gilt oocytes was higher than that from sows. However, the expression of SOD1 and glutathione synthetase (GSS) was higher in the oocytes from sows than in those from gilts. Although DON and BEA impair cell development by diverse mechanisms, this redox imbalance may partially explain the vulnerability of gilt oocytes to these mycotoxins.

Ammonium Ferric Citrate induced Ferroptosis in Non-Small-Cell Lung Carcinoma through the inhibition of GPX4-GSS/GSR-GGT axis activity.

The morbidity and mortality rates associated with non-small-cell lung carcinoma (NSCLC) are increasing every year, placing new demands on existing therapies and drugs. Ammonium ferric citrate (AFC) is often used as a food additive for iron supplementation; however, to our knowledge, no studies have investigated whether AFC can induce ferroptosis in NSCLC. In this study, we demonstrated that specific concentrations of AFC effectively inhibit the proliferation and invasion of lung cancer cell lines in vitro using a cell proliferation inhibition test, a transwell assay, and flow cytometry analysis of cell cycle and apoptosis. In addition, AFC significantly induced oxidative stress injury in lung cancer cell lines. A quantitative polymerase chain reaction assay showed that AFC markedly reduced the expression levels of cell growth factors, negative regulators of ferroptosis, and autophagy regulators. Lastly, a protein-protein interaction analysis revealed that glutathione peroxidase 4 (GPX4) exerted its biological role through the regulation of the GSS/GSR complex and downstream GGT family proteins. When the expression of GPX4 changes, its biological activities, such as the glutathione metabolic process, cellular biosynthetic process, cellular response to chemical stimulus, and antioxidant activity, change accordingly, thereby affecting the survival quality and physiological and biochemical activities of cells. Overall, this study verifies that AFC has the biological activity of activating oxidative stress injury in NSCLC cell lines, leading to a decrease in their autophagy and inducing ferroptosis. We also confirmed that the GPX4-GSS/GSR-GGT axis is a crucial target of AFC-induced ferroptosis.

Programming Integrative Multienzyme Systems and Ionic Strength For Recyclable Synthesis of Glutathione.

In the enzymatic cascade catalysis, it is a big challenge to construct a stable and reusable catalyst with targeted enzymes. The artificial multienzyme reactor has attracted great attention due to its potential for facilitating the performance of enzyme catalysis. In this study, we set up a reliable system that could assemble polyphosphate kinase (PPK) with bifunctional glutathione synthetase (GshF) via SpyCatcher/SpyTag to form multienzyme systems (MESs). Furthermore, MESs could assemble into nanoaggregates by altering the ionic strength, and the larger nanoaggregates could be applied in robust and reusable synthesis of glutathione (GSH). To enhance MES levels in vivo, gene duplication and different coexpression modes were performed. Finally, the optimized production of GSH and oxidized glutathione (GSSG) reached 102.6 and 6.7 mM within 2 h. Compared with the first round, the total yield only decreased by 9.4% after five continuous rounds of biocatalysis.

Individual and Combined Effects of Aflatoxin B1 and Sterigmatocystin on Lipid Peroxidation and Glutathione Redox System of Common Carp Liver.

The purpose of the study was to evaluate the short-term effects of aflatoxin B1 (AFB1 100 µg/kg feed) and sterigmatocystin (STC 1000 µg/kg feed) exposure individually and in combination (100 µg AFB1 + 1000 µg STC/kg feed) on the parameters of lipid peroxidation and glutathione redox system both in biochemical and gene expression levels in one-year-old common carp. Lipid peroxidation parameters were slightly affected, as significant differences were observed only in conjugated diene and triene concentrations. Reduced glutathione content decreased more markedly by STC than AFB1 or AFB1+STC, but glutathione peroxidase activity did not change. Expression of gpx4a, gpx4b, gss, and gsr genes was down-regulated due to STC compared to AFB1 or AFB1+STC, while an induction was found as effect of AFB1+STC in the case of gpx4a, but down-regulation for gpx4b as compared to AFB1. Expression of the glutathione biosynthesis regulatory gene, gss, was higher, but glutathione recycling enzyme encoding gene, gsr, was lower as an effect of AFB1+STC compared to AFB1. These results are supported by the changes in the expression of transcription factors encoding genes, nrf2, and keap1. The results revealed that individual effects of AFB1 and STC on different parameters are synergistic or antagonistic in multi-toxin treatment.

Neurotherapeutic and antioxidant response of D-ribose-L-Cysteine nutritional dietary supplements on Alzheimer-type hippocampal neurodegeneration induced by cuprizone in adult male wistar rat model.

INTRODUCTION: Cuprizone is a neurotoxicant causing neurodegeneration through enzymes inhibition and oxidative stress. D-Ribose-L-Cysteine (DRLC) is a powerful antioxidant with neuroprotective properties. This study explored the antioxidant response of DRLC against cuprizone-induced behavioral alterations, biochemical imbalance and hippocampal neuronal damage in adult wistar rats. MATERIALS AND METHODS: Thirty two (32) adult male wistar rats (150-200g) were divided into four groups (n = 8). Group A received normal saline only as placebo; Group B received 0.5% cuprizone diet only; Group C received a combination of 0.5% cuprizone diet and 100 mg/kg bw of DRLC and Group D received 100 mg/kg bw of DRLC only. The administration was done through oral gavage once daily for 45 days. After the last treatment, neurobehavioral tests (Morris Water Maze and Y maze) was conducted; animals sacrificed and brain harvested for histological analysis and biochemical estimations of levels of antioxidants, oxidative stress markers, neurotransmitters and enzyme activitties. RESULTS: The results showed significant memory decline, hippocampal alterations, decrease levels of antioxidant markers, enzyme and neurotransmitters activities with concomitant increase in norepinephrine and oxidative stress markers in cuprizone induced rats relative to normal but was attenuated with DRLC administration. CONCLUSION: Cuprizone causes cognitive impairment and neurodegeneration through oxidative stress; however, administration of DRLC ameliorated neuropathological alteration induced by cuprizone.

2-Oxothiazolidine-4-carboxylic acid inhibits vascular calcification via induction of glutathione synthesis.

Arterial medial calcification (AMC), the deposition of hydroxyapatite in the medial layer of the arteries, is a known risk factor for cardiovascular events. Oxidative stress is a known inducer of AMC and endogenous antioxidants, such as glutathione (GSH), may prevent calcification. GSH synthesis, however, can be limited by cysteine levels. Therefore, we assessed the effects of the cysteine prodrug 2-oxothiazolidine-4-carboxylic acid (OTC), on vascular smooth muscle cell (VSMC) calcification to ascertain its therapeutic potential. Human aortic VSMCs were cultured in basal or mineralising medium (1 mM calcium chloride/sodium phosphate) and treated with OTC (1-5 mM) for 7 days. Cell-based assays and western blot analysis were performed to assess cell differentiation and function. OTC inhibited calcification ≤90%, which was associated with increased ectonucleotide pyrophosphatase/phosphodiesterase activity, and reduced apoptosis. In calcifying cells, OTC downregulated protein expression of osteoblast markers (Runt-related transcription factor 2 and osteopontin), while maintaining expression of VSMC markers (smooth muscle protein 22α and α-smooth muscle actin). GSH levels were significantly reduced by 90% in VSMCs cultured in calcifying conditions, which was associated with declines in expression of gamma-glutamylcysteine synthetase and GSH synthetase. Treatment of calcifying cells with OTC blocked the reduction in expression of both enzymes and prevented the decline in GSH. This study shows OTC to be a potent and effective inhibitor of in vitro VSMC calcification. It appears to maintain GSH synthesis which may, in turn, prevent apoptosis and VSMCs gaining osteoblast-like characteristics. These findings may be of clinical relevance and raise the possibility that treatment with OTC could benefit patients susceptible to AMC.

Synthetic Virus-like Particles for Glutathione Biosynthesis.

Protein-based nanocompartments found in nature have inspired the development of functional nanomaterials for a range of applications including delivery of catalytic activities with therapeutic effects. As glutathione (GSH) plays a vital role in metabolic adaptation and many diseases are associated with its deficiency, supplementation of GSH biosynthetic activity might be a potential therapeutic when delivered directly to the disease site. Here, we report the successful design and production of active nanoreactors capable of catalyzing the partial or complete pathway for GSH biosynthesis, which was realized by encapsulating essential enzymes of the pathway inside the virus-like particle (VLP) derived from the bacteriophage P22. These nanoreactors are the first examples of nanocages specifically designed for the biosynthesis of oligomeric biomolecules. A dense packing of enzymes is achieved within the cavities of the nanoreactors, which allows us to study enzyme behavior, in a crowded and confined environment, including enzymatic kinetics and protein stability. In addition, the biomedical utility of the nanoreactors in protection against oxidative stress was confirmed using an in vitro cell culture model. Given that P22 VLP capsid was suggested as a potential liver-tropic nanocarrier in vivo, it will be promising to test the efficacy of these GSH nanoreactors as a novel treatment for GSH-deficient hepatic diseases.

Decisive Enzymes and Prediction Models for the Glutathione Content in Spinach (Spinacia oleracea L.) Exposed to Cadmium.

In plants, glutathione (GSH) is crucial for the detoxification and tolerance of heavy metals. However, the change characteristics and decisive enzymes involved in GSH metabolism under heavy metal exposure are still unclear. Based on long-term exposure cultivation of spinach and monitoring of the change trends of enzyme activity and GSH contents in response to cadmium (Cd) stress, these issues were clarified. Spinach goes through three statuses in sequence in response to Cd stress, that is, perception status (PS), response status (RS), and new stable status. With the increase in the Cd concentration, the durations of the PS and RS and the time to reach the peaks in the roots were shorter. However, the durations of the PS and the time to reach the peaks in the leaves were longer. The enzyme activities changed significantly in response to diverse Cd stress in RS. γ-glutamyl transpeptidase was vital to the GSH content in roots. Glutathione synthase was important for the GSH content in leaves. The results of this study provide valuable information to find an efficient way to perform GSH adjustments to fulfill the goal of ensuring food safety.

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.

Solasonine promotes ferroptosis of hepatoma carcinoma cells via glutathione peroxidase 4-induced destruction of the glutathione redox system.

Solasonine is a compound isolated from Solanum melongena that has anti-infection properties, and promotes neurogenesis. However, the use of solasonine for the treatment of hepatocellular carcinoma (HCC) has not yet been reported. So, the aim of this study was to assess the efficacy of solasonine for the treatment of HCC. The effects of solasonine were tested using the HCC cell lines HepG2 and HepRG. Metabolomics analysis was conducted to assess the effects of solasonine on tumor growth of nude mice xenografts using HepG2 cells. The data demonstrated that solasonine significantly suppressed proliferation of HepG2 and HepRG cells. A mouse xenograft model of HepG2 tumor formation confirmed that solasonine suppressed tumor volume and weight, and inhibited HCC cell migration and invasion, as determined with the Transwell and scratch wound assays. To further reveal the underlying regulatory mechanism, metabolomics analysis was performed. The results revealed the effects of solasonine on glutathione metabolism, including glutathione peroxidase 4 (GPX4) and glutathione synthetase (GSS). The glutathione-dependent lipid hydroperoxidase GPX4 prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death. Solasonine increased lipid ROS levels in HepG2 cells by suppression of GPX4 and GSS. However, the use of a ferroptosis inhibitor reversed solasonine-induced ROS production and cell apoptosis. Taken together, these results demonstrate that solasonine promotes ferroptosis of HCC cells via GPX4-induced destruction of the glutathione redox system.

Flavokawain A inhibits prostate cancer cells by inducing cell cycle arrest and cell apoptosis and regulating the glutamine metabolism pathway.

Flavokawain A (FKA), a major chalcone in kava extracts, has exhibited anti-proliferative and apoptotic effects in the prostate cancer. However, the molecular mechanism of FKA remains unclear. In this study, FKA induces cell apoptosis and cell cycle arrest in a G2M phase to prostate cancer cells. FKA interferes with tubulin polymerization and inhibits survivin expression in PC3 cells. Molecular docking simulation experiment finds that FKA can bind to colchicine binding sites that inhibit tubulin polymerization. FKA treatment regulates the glutamine metabolism pathway in PC3 cells by reducing intracellular glutamine, glutamic and proline. FKA treatment also decreases the GSH content by decreasing the activity of GSH synthetase (GSS) and increasing the activity of glutathione thiol transferase (GSTP1), which subsequently induces ROS production and PC3 cell apoptosis.

Effects of Helicobacter pylori on the glutathione-related pathway in gastric epithelial cells.

Virulence factors of Helicobacter pylori (H. pylori) are diverse, so various biological responses happen in a host infected with H. pylori. The aim of this study is to conduct the metabolomics-based evaluation on H. pylori infection. AGS human gastric carcinoma cells were infected with H. pylori strain 26695, and then the altered metabolite pathways in the infected AGS cells were analyzed by metabolomics. Metabolites related to the glutathione (GSH) cycle were downregulated by H. pylori infection. Next, we evaluated the effects of H. pylori on the GSH-related pathway in AGS cells infected with H. pylori isolated from patients with atrophic gastritis (AG), duodenal ulcer (DU) and gastric cancer (GC). We found that the declined degree of GSH levels and oxidative stress were greater in AGS cells infected with GC strains than DU and AG-derived strains. There were no significant differences in almost mRNA expressions of GSH-related factors among different clinical strains, but the protein expression of glutathione synthetase was lower in AGS cells infected with GC-derived strains than DU and AG-derived strains. Our data demonstrates that GC-derived H. pylori-induced oxidative stress in a host is stronger and GC-derived strains may have suppressive influences on the host's GSH-related defense systems.

Vagus Nerve Stimulation Alleviates Hepatic Ischemia and Reperfusion Injury by Regulating Glutathione Production and Transformation.

Inflammation and oxidative stress are pivotal mechanisms for the pathogenesis of ischemia and reperfusion injury (IRI). Vagus nerve stimulation (VNS) may participate in maintaining oxidative homeostasis and response to external stimulus or injury. We investigated whether the in vivo VNS can protect the liver from IRI. In this study, hepatic IRI were induced by ligating the vessels supplying the left and middle lobes of the liver, which underwent 1 h occlusion followed with 24 h reperfusion. VNS was initiated 15 min after ischemia and continued 30 min. Hepatic function, histology, and apoptosis rates were evaluated after 24 h reperfusion. Compared with the IRI group, VNS significantly improved hepatic function. The protective effect was accompanied by a reduction in histological damage in the ischemic area, and the apoptosis rate of hepatocytes has considerable reduction. To find the underlying mechanism, proteomic analysis was performed and differential expression of glutathione synthetase (GSS) and glutathione S-transferase (GST) was observed. Subsequently, test results indicated that VNS upregulated the expression of mRNA and protein of GSS and GST. Meanwhile, VNS increased the plasma levels of glutathione and glutathione peroxidases. We found that VNS alleviated hepatic IRI by upregulating the antioxidant glutathione via the GSS/glutathione/GST signaling pathway.

Arsenic toxicity and its mitigation in ectomycorrhizal fungus Hebeloma cylindrosporum through glutathione biosynthesis.

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.

Egg white consumption increases GSH and lowers oxidative damage in 110-week-old geriatric mice hearts.

The number of geriatrics with an advanced age is rising worldwide, with attendant cardiovascular disorders, characterized by elevated oxidative stress. Such oxidative stress is accelerated by an age-related loss of critical antioxidants like glutathione (GSH) and dietary solutions to combat this loss does not exist. While egg white is rich in sulphur amino acids (AAs), precursors for GSH biosynthesis, whether they can increase sulphur AA in vivo and augment GSH in the aged myocardium remain unclear. We hypothesized that egg white consumption increases GSH and reduces oxidative damage and inflammation in the geriatric heart. To this end, 101-102 week-old mice were given a AIN 76A diet supplemented with either 9% w/w egg white powder or casein for 8 weeks. Subsequent analysis revealed that egg white increased serum sulphur AA and cardiac GSH, while reducing the cysteine carrying transporter SNAT-2 and elevating glutamine transporter ASCT2 in the heart. Increased GSH was accompanied by elevated expression of GSH biosynthesis enzyme glutathione synthase as well as mitochondrial antioxidants like superoxide dismutase 2 and glutathione peroxidase 1 in egg white-fed hearts. These hearts also demonstrated lower oxidative damage of lipids (4-hydroxynonenal) and proteins [nitrotyrosine] with elevated anti-inflammatory IL-10 gene expression. These data demonstrate that even at the end of lifespan, egg whites remain effective in promoting serum sulphur AAs and preserve cardiac GSH with potent anti-oxidant and mild anti-inflammatory effects in the geriatric myocardium. We conclude that egg white intake may be an effective dietary strategy to attenuate oxidative damage in the senescent heart.

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.