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Abdominal aortic aneurysm (AAA) is characterized by structural deterioration of the aortic wall, leading to aortic dilation and rupture. The aim was to compare 183 low molecular weight metabolites in AAA patients and aorta-healthy controls and to explore if low molecular weight metabolites are linked to AAA growth. Blood samples were collected from male AAA patients with fast (mean 3.3 mm/year, range 1.3–9.4 mm/year, n = 39) and slow growth (0.2 mm/year, range −2.6–1.1 mm/year, n = 40), and from controls with non-aneurysmal aortas (n = 79). Targeted analysis of 183 metabolites in plasma was performed with AbsoluteIDQ p180 kit. The samples were measured on a QTRAP 4500 coupled to an Agilent 1260 series HPLC. The levels of only four amino acids (histidine, asparagine, leucine, isoleucine) and four phosphatidylcholines (PC.ae.C34.3, PC.aa.C34.2, PC.ae.C38.0, lysoPC.a.C18.2) were found to be significantly lower (p &lt, 0.05) after adjustment for confounders among the AAA patients compared with the controls. There were no differences in the metabolites distinguishing the AAA patients with slow or fast growth from the controls, or distinguishing the patients with slow growth from those with fast growth. The current study describes novel significant alterations in amino acids and phosphatidylcholines metabolism associated with AAA occurrence, but no associations were found with AAA growth rate.

Acylcarnitines (ACs) have been shown to have a potential to activate pro-inflammatory signaling pathways and to foster the development of insulin resistance. The first task of the current study was to study the full list of ACs (from C2 to C18) in first episode psychosis (FEP) patients before and after antipsychotic treatment. The second task was to relate ACs to inflammatory and metabolic biomarkers established in the same patient cohort as in our previous studies. Serum levels of ACs were determined with the AbsoluteIDQ p180 kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) using the flow injection analysis tandem mass spectrometry ([FIA]-MS/MS) as well as liquid chromatography ([LC]-MS/MS) technique. Identification and quantification of the metabolites was achieved using multiple reactions monitoring along with internal standards. The comparison of ACs in antipsychotic-naïve first-episode psychosis (FEP) patients (N = 38) and control subjects (CSs, N = 37) revealed significantly increased levels of long-chain ACs (LCACs) C14:1 (p = 0.0001), C16 (p = 0.00002), and C18:1 (p = 0.000001) in the patient group. These changes of LCACs were associated with augmented levels of CARN palmitoyltransferase 1 (CPT-1) (p = 0.006). By contrast, the level of short-chain AC (SCAC) C3 was significantly reduced (p = 0.00003) in FEP patients. Seven months of antipsychotic drug treatment ameliorated clinical symptoms in patients (N = 36) but increased significantly their body mass index (BMI, p = 0.001). These changes were accompanied by significantly reduced levels of C18:1 (p = 0.00003) and C18:2 (p = 0.0008) as well as increased level of C3 (p = 0.01). General linear model revealed the relation of LCACs (C16, C16:1, and C18:1) to the inflammatory markers (epidermal growth factor, IL-2, IL-4, IL-6), whereas SCAC C3 was linked to the metabolic markers (leptin, C-peptide) and BMI. FEP was associated with an imbalance of ACs in patients because the levels of several LCACs were significantly higher and the levels of several SCACs were significantly reduced compared with CSs. This imbalance was modified by 7 months of antipsychotic drug treatment, reversing the levels of both LCACs and SCACs to that established for CSs. This study supports the view that ACs have an impact on both inflammatory and metabolic alterations inherent for FEP.

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.

Atopic dermatitis is a chronic inflammatory disease which usually starts in the early childhood and ends before adulthood. However up to 3% of adults remain affected by the disease. The onset and course of the disease is influenced by various genetic and environmental factors. Although the immune system has a great effect on the outcome of the disease, metabolic markers can also try to explain the background of atopic dermatitis. In this study we analyzed the serum of patients with atopic dermatitis using both targeted and untargeted metabolomics approaches. We found the most significant changes to be related to phosphatidylcholines, acylcarnitines and their ratios and a cleavage peptide of Fibrinogen A-α. These findings that have not been reported before will further help to understand this complex disease.

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 well‑defined 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 Wfs1‑deficient 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.

Assessment of oxidative stress is an important but technically challenging procedure in medical and biological research. The reactive oxygen metabolites (d-ROMs) test is a simple assay marketed for analyzing the total amount of hydroperoxides in serum via the Fenton's reaction. Earlier reports have raised a suspicion that a part of the signal detected in the assay comes from sources other than metabolites generated by oxidative stress. The aim of this study was to identify which serum components interfere with the d-ROMs signal. By application of sodium azide, ethylenediaminetetraacetic acid, sodium dodecylsulphate, varying temperature, and spiking endogenous substances we demonstrate that in the case of mammalian sera the assay determines ceruloplasmin (CP) activity with potential interferences from hydroperoxides, iron level, thiols, and albumin. In sera of avian species hydroperoxides contribute more to the test outcome, but the CP part is insensitive to inhibition by azide. In conclusion, this assay has deficiencies in terms of detecting realistic concentrations of hydroperoxides, is mostly measuring CP and is also interfered with other serum components, making it very difficult to interpret in most biological systems.

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.

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 (β-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), and UPF51 (β-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.

Carotenoid and melanin pigments in the plumage of birds are hypothesized to be sensitive to oxidative stress. We manipulated oxidative status of captive greenfinches (Carduelis chloris L.) by the administration of buthionine sulfoximine (BSO), a selective inhibitor of the synthesis of glutathione (GSH), an intracellular antioxidant. Half of the birds in the treated group, as well as in the control group, also received dietary carotenoid (lutein) supplementation. BSO treatment reduced erythrocyte GSH levels and caused oxidative damage as indicated by the increased concentration of plasma malondialdehyde (MDA), an end product of lipid peroxidation. BSO treatment also reduced the brightness (i.e. increased blackness) of the tips of tail feathers grown during the experiment. These results show that a low systemic GSH level is required for development of eumelanin plumage coloration and that such a low GSH level is also potentially dangerous for the organism. Carotenoid supplementation increased plasma carotenoid levels and chroma of the yellow parts of the feathers grown during the experiment. However, carotenoid supplementation did not reduce plasma MDA levels. Manipulation of GSH did not affect plasma carotenoids or carotenoid-based plumage coloration. These findings argue against the antioxidant function of lutein in vivo and carotenoid signaling of antioxidant status. © 2010. Published by The Company of Biologists Ltd. (Less)

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.

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing α-glutamyl residue in place of the γ-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 α-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.

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.

Summary Background The melanocortin system in the skin coordinates pigmentation and immune response and could be implicated in the pathogenesis of vitiligo. Objectives We aimed to analyze changes in expression of genes involved in skin pigmentation (melanocortin system and enzymes involved in melanin synthesis). Methods With quantitative RT-PCR we measured the mRNA expression levels of eight genes from the melanocortin system and two enzymes involved in melanogenesis. RNA was extracted from both lesional and non-lesional skin of vitiligo patients and in non-sun-exposed skin of healthy subjects. Results POMC (proopiomelanocortin) expression was lower in lesional skin compared to non-lesional skin. Expression of melanocortin receptors was increased in unaffected skin of vitiligo patients compared to healthy subjects and decreased in lesional skin compared to uninvolved skin of vitiligo patients, the differences were statistically significant in the cases of MC1R (melanocortin receptor 1) and MC4R (melanocortin receptor 4). TRP1 and DCT genes were down-regulated in lesional skin compared to non-lesional vitiligo skin or skin of healthy controls and up-regulated in uninvolved vitiligo skin compared to healthy control samples. In non-lesional skin, POMC expression was not elevated, possibly indicating that systemic influences are involved in up-regulation of MC receptor genes. Decreased expression of the analyzed genes in the lesional skin is not surprising, but statistically significant increased expression of studied genes in non-lesional skin from vitiligo patients is not described previously. Conclusion In our mind, up-regulation of melanocortin system in non-lesional skin could be systemic compensation to restore normal pigmentation in lesions.

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.

E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

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. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.

Thiol-containing compounds are central actors in many biochemical and pharmacological reactions. The response of cells to any stress (including cell division and apoptosis) involves changes in thiol content as they are consumed to protect cells via different actions (direct modification/regulation of biomolecules, antioxidativity, detoxification, signal transmission). The story of glutathione, the basic intracellular thiol-containing compound, ranges throughout different scientific fields. The importance of this biomolecule is highly impressive. Reduced glutathione (GSH) is a principal actor in many physiological and pharmacological reactions. There are about 60 000 entries under “glutathione” found in the Medline database. The aim of this short review is to characterize glutathione and show that due to an intriguing and multifaceted biofunctionality in the human body this tripeptide itself, as well as its analogues, belongs to the group of molecules looking for broad clinical use (besides being excellent antioxidants). This information might draw more attention to the discovery and investigation of glutathione system supporting/relating substances with a substantial clinical impact.

In this work, we have targeted positions 18-38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24-38 (EC50=70 nM) or 27-38 (EC50=80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Cationic amphiphilic peptides stimulate secretion via a receptor-independent action upon G proteins [1]. We have previously utilized chimeric analogs of mastoparan (MP), including galparan (galanin(1–13)-MP [2]), as molecular probes of secretion [3, 4]. Here, we further resolve the structure-activity relationship of peptidyl secretagogs, including rationally designed chimeric MP analogs. The secretory efficacies of 10 MP analogs were significantly higher than 45 unrelated basic peptides. Comparative studies identified MP analogs that are differential secretagogs for 5-hydroxytryptamine (5-HT) and β-hexosaminidase. Peptide-induced activation of phospholipase D (PLD), an enzyme intimately involved in regulated exocytosis [5], correlated with the secretion of β-hexosaminidase but not 5-HT. Thus, these data indicate that different mechanisms are responsible for the exocytosis of 5-HT and β-hexosaminidase, respectively. Moreover, mastoparan analogs are novel tools for probing the molecular details of exocytosis and other biological phenomena.

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.

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimer's disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Structure-activity relationships for the binding of human alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[Tyr0]-CGRP28-37 (apparent pKBs of 7.49+/-0.25, 5.89+/-0.20, 6.18+/-0.19, 5.85+/-0.19 and 5.25+/-0.07). The SK-N-MC receptor appeared CGRP1-like. On Col 29 cells, only halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (M432) had apparent pKBs of 6.64+/-0.16 and 6.42+/-0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. M433 was almost as potent as halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.

As grain prices rise, the search for alternative glycogenic precursors in animal feed becomes increasingly important, and this study was conducted to determine if the replacement of starch with glycerol, as an alternative glycogenic precursor, affects the milk metabolic profile and milk coagulation ability, and therefore the quality of the milk. Eight primiparous mid-lactation Holstein cows were fed during a replicated 4 × 4 Latin square trial with four different isoenergetic rations: (1) control (T0) fed a total mixed ration (TMR) with barley meal; (2) group T1, decreased barley content, replaced isoenergetically with 1 kg crude glycerol; (3) group T2, the barley meal was replaced with 2 kg of crude glycerol; and (4) group T3 the barley meal was replaced with 3 kg of crude glycerol. Rumen, blood and milk samples were collected at the end of every 21-d treatment period. Rumen samples were analysed for proportion of total volatile fatty acid (VFA), blood samples for insulin and glucose, and milk for metabolites (e.g. citric-acid cycle compounds). The change in glycogenic precursors had a positive effect on rumen VFA proportions; the proportion of propionic acid increased (P P P

The Arabidopsis guard cell anion channel SLAC1 is essential for stomatal closure in response to various endogenous and environmental stimuli. Interestingly, here we reveal an unexpected impairment of slac1 alleles on stomatal opening. We report that mutations in SLAC1 unexpectedly slow stomatal opening induced by light, low CO(2) and elevated air humidity in intact plants and that this is caused by the severely reduced activity of inward K(+) (K(+)(in)) channels in slac1 guard cells. Expression of channels and transporters involved in stomatal opening showed small but significant reductions in transcript levels in slac1 guard cells; however, this was deemed insufficient to explain the severely impaired K(+)(in) channel activity in slac1. We further examined resting cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) and K(+)(in) channel sensitivity to [Ca(2+)](cyt) in slac1. These experiments showed higher resting [Ca(2+)](cyt) in slac1 guard cells and that reducing [Ca(2+)](cyt) to < 10 nM rapidly restored the activity of K(+)(in) channels in slac1 closer to wild-type levels. These findings demonstrate an unanticipated compensatory feedback control in plant stomatal regulation, which counteracts the impaired stomatal closing response of slac1, by down-regulating stomatal opening mechanisms and implicates enhanced [Ca(2+)](cyt) sensitivity priming as a mechanistic basis for the down-regulated K(+)(in) channel activity.

The main goal of the present paper was to examine the influence of the replacement ofγ-Glu moiety toα-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting inα-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 andα-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 byγ-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.

Valproic acid (VLP) is a widely used anticonvulsant and mood-stabilizing drug that relieves the endoplasmic reticulum (ER) stress response, a pathogenetic process related to diabetes. The aim of the present study was to evaluate whether acute valproic acid is able to interfere with glucose intolerance in two different diabetes models: The first model was a Wfs1 mutant mouse with an elevated ER stress response and the second model a streptozocin-induced diabetic mouse. VLP (300 mg/kg, i.p.) was administered to Wfs1 knockout (KO) mice and glucose tolerance test was performed 15 min later. VLP did not have an effect on the course of the glucose tolerance test in wild-type mice, while it did normalize the glucose intolerance in Wfs1 knockout mice. Acute valproic acid also lowered the blood glucose levels in streptozocin-treated mice and potentiated the effect of insulin in these mice. Thus, acute valproic acid is effective in lowering blood glucose levels possibly by potentiating insulin action in both Wfs1 KO mice and in streptozocin-induced type 1 diabetic mice.

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.

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.

This chapter provides a manual for a laboratory-based short course to introduce the common techniques of solid-phase peptide synthesis (SPPS). The course provides students the opportunity to design and manually synthesize analogs of glutathione using relatively simple equipment available in any unsophisticated laboratory. The manual provides compact protocols for both the different steps of SPPS and the final cleavage of peptides from resin supports. We also introduce a simple method for the synthesis of combinatorial libraries of glutathione analogs that is suitable for those relatively unfamiliar with the field of peptide chemistry.

Mastoparan (MP), an amphiphilic tetradecapeptide component of wasp venom, is able to directly activate G proteins and stimulate mast cell degranulation in a receptor-independent manner [1]. Chimeric peptides combine two or more bioactive modules in a single molecule. Previous studies have demonstrated that MP-containing chimeric constructs exhibit G protein-coupled receptor (GPCR)-independent actions dissimilar to those of their individual components [2].

To determine the domains essential for G-protein coupling of the human galanin receptor type 1 (GalR1), we have used both GalR1 mutants and synthetic receptor-derived peptides in(125)I-galanin and [(35)S]-GTPgammaS binding studies. Replacement of potential phosphorylation sites by Leu in the third intracellular loop (IC3) of GalR1 did not affect K(D)values for the receptor. Peptides derived form the IC3 loop, and especially the N-terminal part of it were able to increase the rate of [(35)S]-GTPgammaS binding to the trimeric Gialpha1beta1gamma2, but not to Gsalphabeta1gamma2, whereas the peptides corresponding to the IC1 and IC2 loops had no such effect. IC3 loop peptides also inhibited the binding of(125)I-galanin to GalR1 in membranes from Rin m5F cells. Our results suggest that the IC3 loop of GalR1, especially its N-terminal part, defines the coupling of the receptor to the Gialpha1beta1gamma2 protein and consequently, to the signal transduction cascade.

Ca2+ efflux, Ca(2+)-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca2+ release over a range of concentrations from 0.3-10 microM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca(2+)-ATPases and directly activate the ryanodine receptor (RyR) to release Ca2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP3 receptor (InsP3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca(2+)-mobilising action of MP and chimeric constructs.

Chronic oxidative stress (OS), a major mechanism of chronic obstructive pulmonary disease (COPD), may cause significant damage to DNA. Poly(ADP-ribose) polymerase (PARP)-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR) 7.88, 95% CI 4.26-14.57; p

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

PNA is a nucleic acid analog with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units (figure 1). The purine or pyrimidine bases are linked to the each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid (4). PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules (5-7). The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks (8). These properties of PNA oligomers suggest that they could potentially serve as efficient antisense or antigene reagents. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional (9) and translational level (10,11), and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells (12). However, contrary to the "normal" nucleic acid analogs, PNA oligomers are not efficiently delivered into the cytoplasm of the cell, and until recently this has hindered the application of PNA oligomers as antisense reagents. In this work we summarize some recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.

Cell-penetrating peptides, CPPs, are used as delivery vectors for pharmacologically interesting substances, such as antisense oligonucleotides, proteins and peptides. We present a general principle for designing cell-penetrating peptides derived from naturally occurring proteins as well as from randomly generated polyamino acid sequences. Thereby, we introduce a novel pharmacological principle for identification of cell-penetrating peptides for which the applications can be numerous, including cellular transduction vectors and mimics of intracellular protein–protein interactions. The methods of identifying a CPP comprises assessing the averaged bulk property values of the defined sequence, and ensuring that they fall within the bulk property value interval obtained from the training set. Despite this simplistic approach, the search criteria proved useful for finding CPP properties in either proteins or random sequences. We have experimentally verified cell-penetrating properties of 10–20-mer peptides derived from naturally occurring proteins as well as from random poly-amino acids. We note that since CPPs can be found in part of the protein sequences that may govern protein interactions, it is possible to produce cell-penetrating protein agonists or antagonists.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.