Metabolomics of the Wolfram Syndrome 1 Gene (Wfs1) Deficient Mice.

Wolfram syndrome 1 is a rare autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Mutations in the WFS1 gene encoding the wolframin glycoprotein can lead to endoplasmic reticulum stress and unfolded protein responses in cells, but the pathophysiology at whole organism level is poorly understood. In this study, several organs (heart, liver, kidneys, and pancreas) and bodily fluids (trunk blood and urine) of 2- and 6-month old Wfs1 knockout (KO), heterozygote (HZ), and wild-type (WT) mice were analyzed by untargeted and targeted metabolomics using liquid chromatography-mass spectrometry. The key findings were significant perturbations in the metabolism of pancreas and heart before the onset of related clinical signs such as glycosuria that precedes hyperglycemia and thus implies a kidney dysfunction before the onset of classical diabetic nephropathy. The glucose use and gluconeogenesis in KO mice are intensified in early stages, but later the energetic needs are mainly covered by lipolysis. Furthermore, in young mice liver and trunk blood hypouricemia, which in time turns to hyperuricemia, was detected. In summary, we show that the metabolism in Wfs1-deficient mice markedly differs from the metabolism of WT mice in many aspects and discuss the future biological and clinical relevance of these observations.

Glutathione system in Wolfram syndrome 1­deficient mice.

Wolfram syndrome 1 (WS) is a rare neurodegenerative disease that is caused by mutations in the Wolfram syndrome 1 (WFS1) gene, which encodes the endoplasmic reticulum (ER) glycoprotein wolframin. The pathophysiology of WS is ER stress, which is generally considered to induce oxidative stress. As WS has a well­defined monogenetic origin and a model for chronic ER stress, the present study aimed to characterize how glutathione (GSH), a major intracellular antioxidant, was related to the disease and its progression. The concentration of GSH and the activities of reduction/oxidation system enzymes GSH peroxidase and GSH reductase were measured in Wfs1­deficient mice. The GSH content was lower in most of the studied tissues, and the activities of antioxidative enzymes varied between the heart, kidneys and liver tissues. The results indicated that GSH may be needed for ER stress control; however, chronic ER stress from the genetic syndrome eventually depletes the cellular GSH pool and leads to increased oxidative stress.

Investigation of the surfactant type and concentration effect on the retention factors of glutathione and its analogues by micellar electrokinetic chromatography.

In the present study, a micellar electrokinetic chromatographic method was used to determine the retention factors of hydrophilic monomeric and homodimeric forms of glutathione analogues. Ionic-liquid-based surfactant, 1-tetradecyl-3-methylimidazolium chloride, as well as cetyltrimethylammonium bromide and phosphate buffer (pH 7.4) were employed in the experiments. Since the studied peptides possess a negative charge under physiological conditions, it is expected that the peptides interact with the oppositely charged 1-tetradecyl-3-methylimidazolium chloride and cetyltrimethylammonium bromide micelles via hydrophobically assisted electrostatic forces. The dependence of the retention factor on the micellar concentration of 1-tetradecyl-3-methylimidazolium chloride and cetyltrimethylammonium bromide is nonlinear and the obtained curves converge to a limiting value. The retention factor values of GSH analogues were in the range of 0.36-2.22 for glutathione analogues and -1.21 to 0.37 for glutathione when 1-tetradecyl-3-methylimidazolium chloride was used. When cetyltrimethylammonium bromide was employed, the retention factor values were in the range of 0.27-2.17 for glutathione analogues and -1.22 to 0.06 for glutathione. If sodium dodecyl sulfate was used, the retention factor values of glutathione analogues with carnosine moiety were in the range of -1.54 to 0.38.

Alterations of bronchial epithelial metabolome by cigarette smoke are reversible by an antioxidant, O-methyl-L-tyrosinyl-γ-L-glutamyl-L-cysteinylglycine.

Human bronchial epithelial cells (HBECs) have first-line contact with harmful substances during smoking, and changes in their metabolism most likely represent a defining factor in coping with the stress and development of airway diseases. This study was designed to determine the dynamics of metabolome changes in HBECs treated with cigarette smoke condensate (CSC), and to test whether normal metabolism can be restored by synthetic antioxidants. Principal component analysis, based on untargeted mass spectra, indicated that treatment of CSC-exposed HBECs with O-methyl-L-tyrosinyl-γ-L-glutamyl-L-cysteinylglycine (UPF1) acted faster than did N-acetylcysteine to revert the effect of CSC. The maximum effect of 10 µg/ml CSC itself on HBEC cell line, BEAS-2B, metabolism was seen at 2 hours after treatment, with return to the baseline level by 7 hours. In primary HBECs, the initial maximum effect was seen at 1 hour after CSC exposure. Certain metabolites associated with redox pathways and energy production were affected by CSC. Subsequent restoration of their content by UPF1 supports the hypothetical protective capacity of UPF1 against the oxidative stress and increased energy demand, respectively. Furthermore, UPF1 up-regulated the contents of phospholipid species identified as phosphatidylcholines and phosphatidylethanolamines in the CSC-exposed HBECs, indicating possible suppression of inflammatory processes along with an increase in spermidine as an endogenous cytoprotector. In conclusion, with this dynamic metabolomics study, we characterize the durability of the CSC-induced metabolic changes in BEAS-2B line cells and primary HBECs, and demonstrate the ability of UPF1 to significantly accelerate the recovery of HBECs from CSC insult.

Male infertility: decreased levels of selenium, zinc and antioxidants.

In this study, we aimed to compare the level of zinc, selenium, glutathione peroxidase activity and antioxidant status in following populations of men: severe inflammation in prostate (>10(6) white blood cells in prostate secretion; n=29), severe leukocytospermia, (>10(6) white blood cells in semen; n=31), mild inflammation, (0.2-1M white blood cells in semen or prostate secretion; n=24), non-inflammatory oligozoospermia (n=32) and healthy controls (n=27). Male partners of infertile couples had reduced level of antioxidative activity, selenium and zinc in their seminal plasma. Most importantly, reduced selenium levels were evident in all patient groups regardless of inflammation status. Therefore, these patients might gain some benefit from selenium supplementation.

Separation of glutathione and its novel analogues and determination of their dissociation constants by capillary electrophoresis.

A CE protocol was developed to separate reduced glutathione and its four novel analogues UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), UPF17 (Tyr(Me)-α-Glu-Cys-Gly), UPF50 (ß-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), and UPF51 (ß-Ala-His-Tyr(Me)-α-Glu-Cys-Gly), and their homo- and heterodimers by varying the ionic strength and/or pH of different BGEs. For the determination of dissociation constants (pK(a)) of the above-mentioned peptides the CE method was used. Effective electrophoretic mobilities of analytes were measured in the pH range 5.50-10.00 using optimized BGE with an ionic strength of 50 mM at 25°C. pK(a) values were calculated by fitting the experimental points to a suitable model with correlation coefficients higher than 0.99. The pK(a) values for imidazolyl, amino and thiol moieties of the analyzed peptides were in the range 5.94-6.29, 8.81-9.10, and 7.86-8.13, respectively.

Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1.

Airway epithelium is a principal target for inhaled oxidants like cigarette smoke, which induce epithelial injury and thereby provoke pathogenesis of chronic airway diseases. Alterations in airway epithelial glutathione (GSH) metabolism are central in causing a loss of reducing environment, however, data are scarce on epithelial cells from larger bronchi. We showed a transient depletion of intracellular GSH in human bronchial epithelial cells after exposure to cigarette smoke condensate (CSC), which later followed by a prolonged elevation. Of the GSH-regulating enzymes, CSC increased mRNA expression of both catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase. UPF1, a tetrapeptide GSH analogue, 4-methoxy-L-tyrosinyl-γ-L-glutamyl-L-cysteinyl-glycine, known to possess a 50-fold higher hydroxyl radical scavenging efficiency than does GSH, normalized the intracellular GSH level in the human bronchial epithelial cells under oxidative stress caused by CSC. UPF1 restored the GCLM and GSH reductase mRNA levels, which were significantly augmented by CSC treatment, back to the level of untreated control cells, referring to a successful establishment of control by UPF1 upon the over-accumulation of GSH. Moreover, UPF1 showed a significantly more potent antioxidant capacity than did N-acetyl-L-cysteine (NAC) and, compared to NAC, demonstrated a better potential to assure the whole GSH homeostasis in human bronchial epithelial cells. The current study suggests that UPF1 is capable of maintaining intracellular GSH level under CSC-induced oxidative stress in bronchial epithelial cells via balanced control over GSH-regulating enzymes, reflecting an improved perception of cellular redox conditions and thereby warranting improved adjustment of GSH accumulation.

Mitochondrial oxidative stress index, activity of redox-sensitive aconitase and effects of endogenous anti- and pro-oxidants on its activity in control, Alzheimer's disease and Swedish Familial Alzheimer's disease brain.

Efficient function of the mitochondrial respiratory chain and the citric acid cycle (CAC) enzymes is required for the maintenance of human brain function. A conception of oxidative stress (OxS) was recently advanced as a disruption of redox signalling and control. Mitochondrial OxS (MOxS) is implicated in the development of Alzheimer's disease (AD). Thus, both pro- and anti-oxidants of the human body and MOxS target primarily the redox-regulated CAC enzymes, like mitochondrial aconitase (MAc). We investigated the specific activity of the MAc and MOxS index (MOSI) in an age-matched control (Co), AD and Swedish Familial AD (SFAD) post-mortem autopsies collected from frontal cortex (FC) and occipital primary cortex (OC) regions of the brain. We also examined whether the mitochondrial neuroprotective signalling molecules glutathione, melatonin and 17-ß-estradiol (17ßE) and mitochondrially active pro-oxidant neurotoxic amyloid-ß peptide can modulate the activity of the MAc isolated from FC and OC regions similarly or differently in the case of Co, AD and SFAD. The activity of redox-sensitive MAc may directly depend on the mitochondrial oxidant/antioxidant balance in age-matched Co, AD and SFAD brain regions.

Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562.

The main goal of the present paper was to examine the influence of the replacement of γ-Glu moiety to α-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting in α-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 and α-GSH decreased the activity of CuZnSOD in K562 cells, at peptide concentration of 10 µM by 42% and 38% or 35% and 24%, respectively. After three-hour incubation, UPF1 increased and UPF17 decreased the intracellular level of total GSH. Additionally, it was shown that UPF1 is not degraded by γ-glutamyltranspeptidase, which performs glutathione breakdown. These results indicate that effective antioxidative character of peptides does not depend only on the reactivity of the thiol group, but also of the other functional groups, and on the spatial structure of peptides.

Monitoring the redox cycle of low-molecular peptides using a modified target plate in MALDI-MS.

A new method is being proposed for preparing MALDI target plates with a hydrophobic polymer coating and hydrophilic anchors. The particles of the MALDI matrix were pre-mixed with a poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] solution prior to their placement on a mass-spectrometric sample support. This technique led to the formation of matrix microspots with a diameter of less than 1mm inside the polymer. The polymer and matrix concentration as well as the amount of suspension placed on the target plate influenced the size and quality of microspots to a great extent. The sensitivity of the mass-spectrometric analysis was confirmed by obtaining the mass spectra of fmole concentrations of an apomyoglobin tryptic digest. The potential proteomic application of this type of MALDI surface preparation was demonstrated by performing the redox cycle using glutathione and its analogue. All reactions were carried out directly on a MALDI plate, which accommodates low volumes of reagents and prevents sample loss.

Immunological, antioxidative, and morphological response in combined treatment of ofloxacin and Lactobacillus fermentum ME-3 probiotic in Salmonella Typhimurium murine model.

We aimed to elucidate the immunological (cytokines), biochemical (antioxidative), and patho-morphological responses in the gut and liver evoked by the addition of Lactobacillus fermentum ME-3 to ofloxacin (OFX) treatment in an experimental infection model of Salmonella enterica serovar Typhimurium. After challenge with S. Typhimurium and treatment according to different schemes, either with OFX and/or addition of L. fermentum ME-3, the mice were killed. Blood, liver, spleen, and small intestine samples were plated to detect S. Typhimurium and lactobacilli. Histological slides were prepared from the liver and ileum. The cytokines (IL-10, IFN-γ, and TNF-α), the glutathione peroxidase and reductase, the glutathione ratio, and the lipid peroxides (LPO) in mucosa of the small intestine and liver were estimated. The addition of L. fermentum ME-3 to OFX increased the eradication of S. Typhimurium from tested sites because of antagonistic and antioxidative properties, reduced the presence of typhoid nodules in the liver, and decreased the values of LPO. The immunological response included the reduction of pro-inflammatory cytokines interferon-γ and tumour necrosis factor-α and the increase in anti-inflammatory cytokine interleukin-10 in the livers of mice without typhoid nodules.

Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling.

Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale "omics" studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R(9)) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography-mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R(9) treatment. The cells could recover from a treatment with 5 microM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.

Mechanism and stoichiometry of 2,2-diphenyl-1-picrylhydrazyl radical scavenging by glutathione and its novel alpha-glutamyl derivative.

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing alpha-glutamyl residue in place of the gamma-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.61 mM for glutathione and K = 0.27 mM for the glutathione alpha-glutamyl analog, respectively. The complex formation was followed by a reaction step that was characterized by the similar rate constant k = 0.02 s(-1) for both peptides. Thus, the apparently different antioxidant activity of these two peptides, observed under common assay conditions, was determined by differences in the formation of this non-covalent complex.

Characterization of UPF peptides, members of the glutathione analogues library, on the basis of their effects on oxidative stress-related enzymes.

Previously the authors have designed and synthesized a library of antioxidative glutathione analogues called UPF peptides which are superior to glutathione in hydroxyl radical elimination. This paper is a follow-up study which investigated the effects of the most promising members of the library (UPF1 and UPF17) on oxidative stress-related enzymes. At concentrations used in vivo experiments neither UPF peptide influenced the activity of glutathione peroxidase (GPx) when purified enzyme or erythrocyte lysate was used. At higher concentrations they inhibited GPx activity. UPF peptides had no effect on glutathione reductase (GR) activity. Also they, as well as glutathione itself, slightly increased MnSOD activity in human brain mitochondria and inhibited oxidative burst caused by neutrophil NAD(P)H oxidase. RT-PCR measurements showed that UPF1 and UPF17 have no effect on GPx and MnSOD expression level in human blood mononuclear cells. The results of this study confirm that investigated UPF peptides do not interfere with the enzymatic mechanisms of antioxidative defence and can be used as themselves or as a lead for the protector molecule design against excessive oxidative stress.

Oxidative stress due to (R)-styrene oxide exposure and the role of antioxidants in non-Swiss albino (NSA) mice.

Styrene produces lung and liver damage that may be related to oxidative stress. The purpose of this study was to investigate the toxicity of (R)-styrene oxide (R-SO), the more active enantiomeric metabolite of styrene, and the protective properties of the antioxidants glutathione (GSH), N-acetylcysteine (NAC), and 4-methoxy-L-tyrosinyl-gamma-L-glutamyl-L-cysteinyl-glycine (UPF1) against R-SO-induced toxicity in non-Swiss Albino (NSA) mice. UPF1 is a synthetic GSH analog that was shown to have 60 times the ability to scavenge reactive oxygen species (ROS) in comparison to GSH. R-SO toxicity to the lung was measured by elevations in the activity of lactate dehydrogenase (LDH), protein concentration, and number of cells in bronchoalveolar lavage fluid (BALF). Toxicity to the liver was measured by increases in serum sorbitol dehydrogenase (SDH) activity. Antioxidants were not able to decrease the adverse effects of R-SO on lung. However, NAC (200 mg/kg) ip and GSH (600 mg/kg), administered orally prior to R-SO (300 mg/kg) ip, showed significant protection against liver toxicity as measured by SDH activity. Unexpectedly, a synthetic GSH analog, UPF1 (0.8 mg/kg), administered intravenously (iv) prior to R-SO, produced a synergistic effect with regard to liver and lung toxicity. Treatment with UPF1 (0.8 mg/kg) iv every other day for 1 wk for preconditioning prior to R-SO ip did not result in any protection against liver and lung toxicity, but rather enhanced the toxicity when administered prior R-SO. The results of the present study demonstrated protection against R-SO toxicity in liver but not lung by the administration of the antioxidants NAC and GSH.

Design, synthesis and properties of novel powerful antioxidants, glutathione analogues.

Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC(50) of GSH: 1,231.0 +/- 311.8 microM; EC(50) of UPF peptides: from 0.03 to 35 microM) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC(50): 11.4 +/- 1.0 microM). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 microM concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.

Comparison of the stability of glutathione and related synthetic tetrapeptides by HPLC and capillary electrophoresis.

Glutathione and related peptides are interesting targets as protectors of biological systems against an oxidative injury. Two novel glutathione analogues, UPF1 and UPF15, have been designed and synthesised. As a result of different reactions taking place, the thiol-containing compounds oxidise to disulfides. In this study, the stability of UPF1, UPF15 and glutathione in various solutions was investigated by using HPLC and CE. The results showed that UPF1 and UPF15 are powerful hydroxyl radical scavengers and their dimerisation process velocity is higher than that of glutathione.

Characterization of the antioxidative activity of novel nontoxic neuropeptides by using capillary electrophoresis.

In the present study, we have monitored the oxidation process of novel nontoxic neuropeptides and determined its rate constants, which describe the antioxidative potential of the peptides. A capillary electrophoretic method was implemented which ensures the simultaneity of analysis of reactants and products in a short time of analysis. The rate constants of oxidation of the four novel peptides, 4-methoxy-L-tyrosinyl-gamma-L-glutamyl-L-cysteinyl-glycine (UPF1), D-serinyl-gamma-L-glutamyl-L-cysteinyl-glycine (UPF6), 4-methoxy-L-tyrosinyl-alpha-L-glutamyl-L-cysteinyl-glycine and D-serinyl-alpha-L-glutamyl-L-cysteinyl-glycine, designed by us, were compared with those of oxidation of glutathione (reduced glutathione) by using capillary electrophoresis. The second-order rate constants were similar for all peptides if the oxidation was carried out with hydrogen peroxide (k(II) = 0.208 - 0.236 x 10(3)/M.min). The rate constants were also determined for the mixtures of peptides. When the oxidation is caused by hydroxyl radical (OH*), the gamma-glutamate containing peptides (UPF1 and UPF6) exhibited two to four times higher antioxidative activity (k(II) = 4.428 and 2.152 x 10(3)/M.min, respectively). The results suggest that the antioxidative potential of the peptides studied is not determined by the formation of disulphide bridge alone.

Amyloid precursor protein carboxy-terminal fragments modulate G-proteins and adenylate cyclase activity in Alzheimer's disease brain.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer's disease (AD) and age-matched control brains was compared. 10 microM APP(639-648)-APP(657-676) (PEP1) causes a fivefold stimulation in the [35S]GTPgammaS-binding to control hippocampal G-proteins. APP(657-676) (PEP2) and APP(639-648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649-669) (PEP3) showed no regulatory activity in the [35S]GTPgammaS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [35S]GTPgammaS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

Anticonvulsant activity of a nonpeptide galanin receptor agonist.

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace (125)I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace (125)I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.