Academic literature on the topic 'Advanced oxidation protein product'

Glycation and subsequent Maillard or browning reactions of glycated proteins, leading to the formation of advanced glycation end products (AGEs), are involved in the chemical modification of proteins during normal aging and have been implicated in the pathogenesis of diabetic complications. Oxidative conditions accelerate the browning of proteins by glucose, and AGE proteins also induce oxidative stress responses in cells bearing AGE receptors. These observations have led to the hypothesis that glycation-induced pathology results from a cycle of oxidative stress, increased chemical modification of proteins via the Maillard reaction, and further AGE-dependent oxidative stress. Here we show that the preparation of AGE-collagen by incubation with glucose under oxidative conditions in vitro leads not only to glycation and formation of the glycoxidation product Nε-(carboxymethyl)lysine (CML), but also to the formation of amino acid oxidation products on protein, including m-tyrosine, dityrosine, dopa, and valine and leucine hydroperoxides. The formation of both CML and amino acid oxidation products was prevented by anaerobic, anti-oxidative conditions. Amino acid oxidation products were also formed when glycated collagen, prepared under anti-oxidative conditions, was allowed to incubate under aerobic conditions that led to the formation of CML. These experiments demonstrate that amino acid oxidation products are formed in proteins during glycoxidation reactions and suggest that reactive oxygen species formed by redox cycling of dopa or by the metal-catalysed decomposition of amino acid hydroperoxides, rather than by redox activity or reactive oxygen production by AGEs on protein, might contribute to the induction of oxidative stress by AGE proteins.

Chronic kidney disease (CKD) has a high prevalence and low cure rate and represents a significant health issue. Oxidative stress is common in CKD due to metabolic disorders, inflammation, and impaired renal function changing normal proteins into advanced oxidation protein products (AOPPs). Huang Gan formula (HGF) is a new type of traditional Chinese herbal medicine. Although we previously investigated the protective effects of HGF against oxidative stress, the mechanism of HGF in CKD is still not fully understood. In this study, we used western blotting, quantitative polymerase chain reaction, and biochemical assays to show that HGF significantly decreased AOPP-induced oxidative stress damage. Moreover, the protective effects of HGF might be associated with upregulation of the advanced glycation end product receptor 1 (AGE-R1) and downregulation of the receptor for advance glycation end products (RAGE). Treatment with HGF and the Janus kinase 2 (JAK2) inhibitor, AG4-90, significantly attenuated AOPP-induced JAK2/STAT3 protein levels. These findings indicate that HGF inhibits AOPP-mediated biological responses by inactivating the JAK2/STAT3 pathway. In conclusion, HGF eliminated AOPP-induced effects in human mesangial cells (HMCs) by interrupting JAK2/STAT3 signaling, which altered RAGE/AGE-R1 expression and reduced oxidative stress in CKD.

Thermal treatments of dairy products favor oxidations, Maillard reactions, and the formation of sugar or lipid oxidation products. Additives including flavorings might enhance these reactions or even induce further reactions. Here we aimed to characterize protein modifications in four flavored milk drinks using samples along the production chain—raw milk, pasteurization, mixing with flavorings, heat treatment, and the commercial product. Therefore, milk samples were analyzed using a bottom up proteomics approach and a combination of data-independent (MSE) and data-dependent acquisition methods (DDA). Twenty-one small carbonylated lipids were identified by shotgun lipidomics triggering 13 protein modifications. Additionally, two Amadori products, 12 advanced glycation end products (AGEs), and 12 oxidation-related modifications were targeted at the protein level. The most common modifications were lactosylation, formylation, and carboxymethylation. The numbers and distribution of modification sites present in raw milk remained stable after pasteurization and mixing with flavorings, while the final heat treatment significantly increased lactosylation and hexosylation in qualitative and quantitative terms. The processing steps did not significantly affect the numbers of AGE-modified, oxidized/carbonylated, and lipid-carbonylated sites in proteins.

Crohn’ disease (CD) patients are at high risk of postoperative recurrence and new tools for the assessment of disease activity are needed to prevent long-term complications. In these patients, the over-production of ROS generated by inflamed bowel tissue and inflammatory cells activates a pathogenic cascade that further exacerbates inflammation and leads to increased oxidative damage to DNA, proteins, and lipids. We measured the products of protein/lipid oxidation and the total antioxidant capacity (ferric reducing ability of plasma, FRAP) in the serum of CD patients with severe disease activity requiring surgery with the aim to characterize their redox status and identify associations between oxidative stress-related markers and their clinical characteristics. At the systemic level, CD was associated with increased levels of protein and lipid oxidation products when compared to healthy volunteers, even though the FRAP values were similar. Advanced oxidation protein product (AOPP) levels showed the highest difference between patients and the controls (11.25, 5.02–15.15, vs. 1.36, 0.75–2.70, median, interquartile range; p < 0.0001) and the analysis of receiver operating characteristic (ROC) curves, indicated for AOPP, the best area under the curve (AUC) value for CD prediction. Advanced glycated end-products (AGEs) were also significantly higher in CD patients (p < 0.01), which is of interest since AOPP and AGEs are both able to activate the membrane receptor for advanced glycation end products (RAGE) involved in inflammatory diseases. Thiobarbituric acid reactive substance (TBARS) levels were significantly higher in CD patients with ileal localization and aggressive disease behavior, in smokers, and in patients suffering from allergies. In conclusion, our data indicate that circulating oxidative stress biomarkers may be attractive candidates as disease predictors as well as for clinical or therapeutic monitoring of CD. Our results also suggest that AOPP/AGEs and RAGE signaling may represent a pathogenic factor and a potential therapeutic target in CD.

Oxidative stress and inflammation cannot be considered as diseases themselves; however, they are major risk factors for the development and progression of the pathogenesis underlying many illnesses, such as cancer, neurological disorders (including Alzheimer’s disease and Parkinson’s disease), autoimmune and metabolic disorders, etc. According to the results obtained from extensive studies, oxidative stress–induced biomolecules, such as advanced oxidation protein products, advanced glycation end products, and advanced lipoxidation end products, are critical for an accelerated level of inflammation and oxidative stress–induced cellular damage, as reflected in their strong affinity to a wide range of scavenger receptors. Based on the limitations of antioxidative and anti-inflammatory molecules in practical applications, targeting such interactions between harmful molecules and their cellular receptors/signaling with advances in gene engineering technology, such as CRISPR or TALEN, may prove to be a safe and effective alternative. In this review, we summarize the findings of recent studies focused on the deletion of scavenger receptors under oxidative stress as a development in the therapeutic approaches against the diseases linked to inflammation and the contribution of advanced glycation end products (AGEs), advanced lipid peroxidation products (ALEs), and advanced oxidation protein products (AOPPs).

IntroductionOxidative stress plays a role in producing advanced oxidation protein products (AOPP). High level of AOPP was observed in patients with such pathological conditions as ischemic heart disease, diabetes, cancer or neurodegenerative diseases.ObjectivesA role of oxidative stress in depression and in antidepressant treatment has been implicated. There have been no studies so far on AOPP in psychiatric diseases.Aim: An assessment of AOPP concentration, as a marker of oxidative stress, in patients with depression and the effect of antidepressant treatment.MethodsThirty-one patients hospitalized at Department of Adult Psychiatry, Poznan University of Medical Sciences, were studied. The first depressive episode was diagnosed in 5 patients, recurrent depressive disorder in 6 patients, and depression in the course of bipolar affective disorder in 20 patients. Patients were treated with venlafaxine (10), paroxetine (7), fluoxetine (5), clomipramine (4), citalopram (3), sertraline (1) and mianserine (1). Advanced oxidation protein products (AOPP) levels were measured twice: before treatment and in remission on maintenance doses of drugs. Control group consisted of 18 healthy volunteers, age- and gender matched.ResultsThere was no significant difference between depressed patients and healthy controls in the AOPP concentration before treatment. There was no correlation between AOPP levels and diagnosis, duration of illness, duration of the current episode and the age of illness’ onset. After antidepressant treatment, a significant decrease of AOPP concentration was found.ConclusionsThe results of the study may confirm previous data suggesting a decrease of some markers of oxidative stress after antidepressant treatment.

The aim of this study was to characterize markers of oxidation of proteins in cattle, developing the assay method of AOPP (advanced oxidation protein products). Using standard commercial proteins (Bovine serum Albumin, bovine g-Globuline), oxidations In vitro were performed with chlorinated oxidant (hypochlorous acid) and hydroperoxide (cumene hydroperoxide) determining the relationship between AOPP and carbonyl groups by spectrophotometric and Western blotting analysis. The conformational changes of the standards were observed by one-dimensional electrophoresis kept in non-reducing conditions. In the second part of this project, we have studied the relationship between inflammation indicators and AOPP in both healthy cow and in animals with inflammatory processes, supporting the hypothesis that AOPP are specific indicators of protein oxidation by chlorinated oxidants produced by neutrophilis. The third part of the work has focused on the production of oxidative standard protein (AOPP-BSA) for the development of ELISA systems to detect autoantibodies directed against epitopes of oxidized proteins.
Lo scopo del presente studio è stato quello di caratterizzare indicatori dell’ossidazione delle proteine nella specie bovina, sviluppando la metodica del dosaggio delle AOPP (advanced oxidation protein products). A partire da standard proteici commerciali (Albumina bovina e g-Globuline bovine) sono state eseguite delle ossidazioni in vitro con ossidante clorurato (acido ipocloroso) ed idroperossido (Cumene idroperossido) determinando la relazione tra AOPP e gruppi carbonilici tramite analisi spettrofotometriche e densitometriche tramite Western blotting. Le modificazioni conformazionali degli standard sono state osservate mediante elettroforesi monodimensionali mantenute in condizioni non riducenti. La seconda parte del progetto sono state studiate le relazioni tra indicatori del processo infiammatorio e le AOPP in animali sani e/o con processi infiammatori in atto, avvalorando l’ipotesi che le AOPP siano degli indicatori specifici dell’ossidazione proteica da parte di ossidanti clorurati di origine neutrofilica. La terza parte del lavoro si è focalizzata sulla produzione di standard proteici ossidati (AOPP-BSA) per la messa a punto di sistemi ELISA per l’individuazione di autoanticorpi diretti contro epitopi ossidati delle proteine.

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Oxidative stress is characterized by an imbalance between the production of free radicals, particularly reactive oxygen species (ROS), and the defense capacity of the organism against these species, leading to a progressive oxidative damage. Proteins are considered to be the primary target for oxidative damage, since they are major components of biological systems and may neutralize 50 to 75% of free radicals. Recently, a new class of compounds formed as a result of oxidative stress was described, referred to as advanced oxidation protein products (AOPP). The accumulation of AOPP was first described in patients with chronic renal failure on hemodialysis and subsequently it was found that this marker is involved in a number of pathological conditions such as diabetes, atherosclerosis, obesity, and acute renal failure. Previous studies have identified AOPP as a new marker of oxidative damage to proteins and a new class of inflammatory mediators promoting effects both at the cellular level, as at systemic level. In this context, different biological structures, including plasma proteins such as albumin and fibrinogen are susceptible to oxidation by ROS. It is known that albumin is the major target of oxidative stress in plasma of uremic patients, however, it has been demonstrated that the fibrinogen is also capable of undergoing oxidative modification. Whereas the oxidative and inflammatory processes are involved in the pathophysiology of a number of clinical conditions and AOPP is a biomarker which can reflect these changes, it is extremely important to evaluate the susceptibility of other proteins to the formation of these products, in addition to albumin. Thus, the aim of this study was to investigate the formation of AOPP from the fibrinogen in an in vitro model and evaluate structural and functional changes in the molecule of this pro-coagulant protein. Thus, to promote in vitro AOPP, fibrinogen was exposed to hypochlorous acid (HOCl) at various concentrations (1, 2 and 4 mM). After checking the effectiveness of fibrinogen to produce AOPP, was demonstrated that the formation of these products promotes functional alterations in fibrinogen, causing changes in their structural domains and increasing their procoagulant activity. Therefore, the fibrinogen can be considered a source of AOPP formation and deterioration caused by this process in the molecule of this protein, may be related to several pathological conditions involving coagulation system and contribute especially in the development of thrombotic processes.
O estresse oxidativo é caracterizado pelo desequilíbrio entre a produção de radicais livres, em particular espécies reativas de oxigênio (EROs), e a capacidade de defesa do organismo contra essas espécies, levando a um progressivo dano oxidativo. As proteínas são consideradas o principal alvo para o dano oxidativo, uma vez que estas são as maiores componentes dos sistemas biológicos e podem neutralizar 50 a 75% dos radicais livres. Recentemente, foi descrita uma nova classe de compostos formados em consequência do estresse oxidativo, designada como produtos proteicos de oxidação avançada (AOPP). O acúmulo de AOPP foi primeiramente descrito em pacientes com insuficiência renal crônica submetidos à hemodiálise e, posteriormente, verificou-se que este marcador está envolvido em uma série de condições patológicas, como diabetes, aterosclerose, obesidade e insuficiência renal aguda. Estudos prévios têm identificado AOPP como um novo marcador de dano oxidativo a proteínas e uma nova classe de mediadores inflamatórios, promovendo efeitos tanto a nível celular, quanto a nível sistêmico. Neste contexto, diferentes estruturas biológicas, incluindo proteínas plasmáticas como a albumina e o fibrinogênio, são passíveis a oxidação por EROs. Sabe-se que a albumina é o principal alvo do estresse oxidativo no plasma de pacientes urêmicos, no entanto, já foi demonstrado que o fibrinogênio também é passível de sofrer modificações oxidativas. Considerando que os processos inflamatórios e oxidativos estão envolvidos na fisiopatologia de uma série de condições clínicas e que AOPP é um biomarcador que pode refletir essas alterações, é de extrema relevância a avaliação da susceptibilidade de outras proteínas à formação desses produtos, além da albumina. Assim, o principal objetivo deste estudo foi investigar a formação de AOPP a partir do fibrinogênio em um modelo in vitro, bem como avaliar alterações estruturais e funcionais na molécula desta proteína pró-coagulante. Desse modo, para a promoção de AOPP in vitro, o fibrinogênio foi exposto ao ácido hipocloroso (HOCl) em diversas concentrações (1, 2 e 4mM). Após a verificação da efetividade do fibrinogênio em produzir AOPP, foi demonstrado que a formação destes produtos promove alterações funcionais no fibrinogênio, causando modificações em seus domínios estruturais e aumentando sua atividade pró-coagulante. Portanto, o fibrinogênio pode ser considerado uma fonte de formação de AOPP e as alterações provocadas por este processo na molécula desta proteína, podem estar relacionadas a diversas condições patológicas envolvendo o sistema da coagulação e contribuir especialmente, no desenvolvimento de processos trombóticos.

The aim of the present study was to evaluate the relationship between AOPP (Advanced oxidation protein products) and bovine neutrophils 'in vitro'. For this purpose AOPP were produced "in vitro" by oxidizing bovine serum albumin with HOCl (hypochlorous acid) and bovine neutrophils were isolated from whole blood of dairy cattles. AOPP-BSA were incubated with freshly isolated bovine neutrophils, unstimulated and stimulated with PMA a strong activator of the respiratory burst. Neutrophils ROS production and viability were measured by luminol-amplified chemiluminescence and by MTT and lactate dehydrogenase assays (LDH), respectively. Results obtained have shown that AOPP-BSA are able to reduce significantly ROS production of PMA stimulated neutrophil and their viability measured by MTT assay, no cell lysis was detected by LDH assay. On the basis of these results, our work has studied if AOPP are able to trigger apoptotic events. For this purpose, caspase 8-9-3 and DNA laddering were used as markers in order to discriminate between the 'intrinsic' and the 'extrinsic' pathway of apoptosis. The results obtained showed that un-stimulated bovine neutrophils incubated with AOPP-BSA show a higher but not significant production of active caspase 8 in comparison with the incubation with BSA. Also caspase 3 display an increase, but not significant, in un-stimulated neutrophils after 6 hours of incubation with AOPP-BSA, respects the incubation with BSA. No differences were obtained for caspase 9 and for DNA laddering. Therefore, in these experimental conditions is possible to conclude that the 'intrinsic' pathway of apoptosis was not involved in the reduced functionality of neutrophils or in their reduced viability, but bovine neutrophils incubated with AOPP-BSA seem to be "accompanied" to the early phases of the 'extrinsic' pathways of apoptosis. In addition, the present work wanted to evaluate the capacity of triggered neutrophils to generate AOPP in vitro. BSA was incubated with un-stimulated and PMA-stimulated bovine neutrophils for 1-2-3 hours and the production of specific markers of protein oxidation such as AOPP, dityrosines and carbonyls was assessed. BSA incubated with stimulated neutrophils presents a significant higher level of AOPP and dityrosines respects the incubation with un-stimulated neutrophils. Carbonyls don't seem to be produced in these condition, at least at the beginning of the incubation. In parallel, BSA incubated with the same concentration of HOCl produced by PMA-stimulated neutrophils, for 1-2-3 hours, presents a higher level of AOPP, dityrosines and carbonyls. Therefore, it's possible to conclude that bovine neutrophils are able to oxidize BSA in vitro and generate chemical and structural modification such as AOPP and dityrosines, in the experimental condition used. However, carbonyls seem to be a non-specific indicator of neutrophils-mediated protein oxidation. The direct exposure of BSA to HOCl couldn't fully mimic the complex events leading to BSA oxidation and AOPP production by activated neutrophils.
Lo scopo del presente studio è stato quello di valutare le relazioni tra AOPP (prodotti avanzati di ossidazione proteica) e i neutrofili di bovino "in vitro". A questo scopo le AOPP sono state generate "in vitro", ossidando l'albumina sierica bovina con HOCl (acido ipocloroso) mentre i neutrofili di bovino sono stati isolati da sangue intero di bovine da latte. Le AOPP-BSA sono state incubate con i neutrofili di bovino appena isolati in condizioni di assenza di stimolo o stimolati con PMA un forte attivatore del "burst" respiratorio. La produzione di ROS da parte dei neutrofili e la loro vitalità , sono state misurate rispettivamente mediante chemiluminescenza amplificata dal luminolo e dai saggi MTT e lattato deidrogenasi (LDH). I risultati ottenuti hanno mostrato che le AOPP-BSA sono in grado di ridurre significativamente la produzione di ROS da parte dei neutrofili stimolati con PMA e la loro vitalità , misurata con il saggio MTT mentre non è stata rilevata lisi cellulare mediante saggio LDH. Sulla base di questi risultati il presente lavoro si è proposto di studiare se le AOPP sono in grado di scatenare eventi apopotici. A questo scopo le caspasi 3, 8 , 9 e la frammentazione del DNA sono stati utilizzati come marker con l'obiettivo di discriminare tra la via intrinseca e quella estrinseca di apoptosi. I risultati ottenuti hanno mostrato che i neutrofili di bovino non stimolati e incubati con AOPP-BSA per 1 ora e 6 ore, presentano una maggiore ma non significativa produzione di caspasi 8 attiva, se comparati con l'incubazione con BSA. Anche la caspasi 3 mosta un incremento, non significativo in neutrofili non stimolati incubati con AOPP-BSA per 6 ore, rispetto all'incubazione con BSA. Non è stata ottenuta alcuna differenza per quanto riguarda la caspasi 9 e la frammentazione del DNA. Tuttavia, in queste condizioni sperimentali è possibile concludere che la via intrinseca dell'apoptosi non è coinvolta nella riduszione della funzionalità  dei neutrofili di bovino o nella loro vitalità  ma i neutrofili di bovino incubati con AOPP-BSA sembrano piuttosto essere 'accompagnati' verso le fasi precoci della via estrinseca dell'apoptosi. Inoltre, il seguente studio ha voluto valutare la capacità  dei neutrofili di bovino attivati di generare AOPP 'in vitro'. La BSA è stata incubata con neutrofili di bovino non stimolati e stimolati con PMA per 1-2-3 ore, ed è stata misurata la formazione di specifici marcatori di ossidazione proteica come le AOPP le ditirosine e i carbonili. La BSA incubata con neutrofili stimolati con PMA, presenta un livello significativamente alto di AOPP e ditirosine rispetto all'incubazione con neutrofili non stimolati. I carbonili invece sembrano non essere prodotti in queste condizioni, almeno nelle fasi inziali dell'incubazione. In parallelo, la BSA incubata con la stessa concentrazione di HOCl prodotta dai neutrofili stimolati, per 1-2-3 ore, presenta livelli più elevato di AOPP, ditirosine e carbonili. Tuttavia è possibile concludere che i neutrofili di bovino sono in grado di ossidare la BSA e generare modificazioni chimiche e strutturali come AOPP e ditirosine nelle condizioni sperimentate. I carbonili invece sembrano non essere un marcatore specifico di ossidazione proteica mediata dai neutrofili. In aggiunta la diretta esposizione della BSA all'HOCl non è in grado di mimare completamente la complessità  degli eventi che portano all'ossidazione della BSA e alla produzione di AOPP da parte dei neutrofili attivati.

CKD is diagnosed when there’s a decreased kidney function shown by a GFR less than 60 ml / min (established for a reference man with 1.73 m² body surface area), or markers of kidney damage, or both, of at least 3 months duration. The severity of complications increases in parallel with the GFR decline. We focused on three comorbidities extremely common in CKD patients: oxidative stress and inflammation; anemia and dysbiosis. We investigated these CKD comorbidities both with in vitro and in vivo approaches. More in detail, regarding in vivo studies, we measured oxidative stress biomarkers in a population of ESRD patients before and after the hemodialysis treatment, comparing the results with a population of healthy subjects; we evaluated oxidative stress biomarkers in the plasma of HD patients before, during and after two type of iron treatments (intravenous and sucrosomial iron). Regarding in vitro experiments, we focused on two uremic toxins, urea and indoxyl sulphate, and we evaluated their effects on a human endothelial cell line (Human Microvascular Endothelial Cells 1, HMEC-1).

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The assessment of biomarkers of reactions involving reactive oxygen species have the potential not only to determine the extent of oxidative damage, but also to predict the effectiveness of therapeutic strategies aimed at reducing or preventing the damage promoted by oxidative stress. Recently, it has been described and characterized a new class of compounds formed in consequence of oxidative stress, designated as advanced oxidation protein products (AOPP). The accumulation of AOPP was first described in patients with chronic renal failure undergoing hemodialysis and was subsequently found in diabetes, atherosclerosis, obesity and acute renal failure. Previous studies have identified AOPP as a new marker of oxidative damage to proteins and a new class of inflammatory mediators, providing arange of effects at both the cellular and systemic levels. Although the mechanism of action by which AOPP act is not fully understood, it is known that these products activate respiratory burst in phagocytes, including neutrophils and monocytes, through the activation of enzymes present in these cells. Furthermore, it has been demonstrated that AOPP may promot these effects (pro-oxidants and pro-inflammatory) at several cell types such as endothelial and kidney cells via activation of a signaling cascade, and in some aspects of this cascade AOPP effects is very similar to effects caused by advanced glycation end products (AGEs). In this context, the evaluation of the antioxidant activity of compounds in vitro models involving the formation of AOPP may present special interest. Among these compounds, N-acetylcysteine (NAC) and Fructose-1 ,6-bisphosphate (FBP) may be promising substances for this purpose. The NAC is a sulfhydryl donor group very similar to the amino acid cysteine and FBP is a highly energetic intermediate metabolite of glycolysis. Thus, the aim of this study was to determine the effects of FBP and NAC, as well as the synergistic effect of both treatments on the formation of AOPP in vitro. For this purpose, purified human albumin was incubated with various concentrations of hypochlorous acid (HOCl) (1, 2 and 4 mM) to produce AOPP in vitro, which was named albumin-advanced oxidation protein products (albumin-AOPP). In this context, both FBP as NAC were able to inhibit the formation of AOPP concentration-dependent manner, with FBP 20mg/mL and NAC 1mg/mL were responsible for the inhibition of 64% and 85% respectively. Furthermore, the synergistic effect promoted by the association of both compounds was more effective ininhibiting the formation of AOPP. Therefore, FBP and NAC may be promising candidates to mitigate or neutralize the pro-inflammatory and pro-oxidant triggered by AOPP.
A avaliação de biomarcadores das reações que envolvem as espécies reativas de oxigênio têm potencial não apenas de determinar a extensão do dano oxidativo, mas também de predizer a eficiência das estratégias terapêuticas destinadas a reduzir ou prevenir os danos promovidos pelo estresse oxidativo. Recentemente, foi descrita e caracterizada uma nova classe de compostos formados em consequência do estresse oxidativo, designada como produtos proteicos de oxidação avançada (AOPP). O acúmulo de AOPP foi primeiramente descrito em pacientes com insuficiência renal crônica submetidos à hemodiálise e, posteriormente, verificou-se que este marcador está envolvido em várias condições patológicas, incluindo diabetes, aterosclerose, obesidade e insuficiência renal aguda. Estudos prévios têm identificado AOPP como um novo marcador de dano oxidativo a proteínas e uma nova classe de mediadores inflamatórios, promovendo uma série de efeitos tanto a nível celular quanto a nível sistêmico. Embora o mecanismo de ação pelo qual os AOPP agem não está totalmente esclarecido, sabe-se que estes produtos ativam o burst respiratório em fagócitos, incluindo neutrófilos e monócitos, através da ativação de complexos enzimáticos presentes nestas células. Além disso, tem sido demonstrado que os AOPP também podem promover efeitos deletéreis (pró-oxidantes e pró-inflamatórios) a vários tipos celulares, como células renais e endoteliais, através da ativação de uma cascata de sinalização, sendo em alguns aspectos desta cascata muito semelhante aos efeitos promovidos pelos produtos finais de glicação avançada (AGEs). Neste contexto, a avaliação da atividade antioxidante e antiinflamaória de compostos em modelos in vitro envolvendo a formação de AOPP pode apresentar especial interesse. Dentre esses compostos, a N-acetilcisteína (NAC) e a Frutose- 1,6-bisfosfato (FBP) podem ser substâncias promissoras para esta finalidade. A NAC é um doador de grupo sulfidrila muito semelhante ao aminoácido cisteína e a FBP é um açúcar bifosforilado e um metabólito intermediário altamente energético da glicólise. Assim, o principal objetivo deste estudo foi determinar o efeito da FBP e da NAC, bem como o efeito sinérgico de ambas, sobre a formação de AOPP in vitro. Para isso, a albumina purificada humana foi incubada com várias concentrações de ácido hipocloroso (HOCl) (1, 2 e 4 mM) para produzir AOPP in vitro, a qual foi denominada de albumina-produtos proteicos de oxidação avançada (albumina-AOPP). Neste contexto, tanto FBP quanto NAC foram capazes de inibir a formação de AOPP de maneira concentração-dependente, sendo que FBP 20 mg/mL e NAC 1mg/mL foram responsáveis pela inibição de 64% e 85% respectivamente. Além disso, o efeito sinérgico promovido pela associação de ambos os compostos foi maisefetivo em inibir a formação de AOPP quando comparado com o efetio promovido pelos compostos isoladamente. Portanto, FBP e NAC podem ser candidatos promissores para amenizar ou neutralizar os efeitos pró-inflamatórios e pró-oxidantes desencadeados pelos AOPP.

Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune disease, characterized by peripheral and symmetrical polyarthritis that leads to joint destruction and deformity due to erosion of cartilage and bone. This is a common disease that affects approximately 1% of the world population and is more common in women than men, however, its peak incidence occurs between the fourth and sixth decades of life. Large amounts of reactive oxygen species (ROS) have been identified in the synovial fluid of RA patients, and such large amounts may lead to oxidative damage of hyaluronic acid, lipid, cartilage matrix and DNA. The accumulation of ROS in the cells, also serves as major intracellular signaling molecules that amplify the inflammatory response synovium proliferative. The objective of this study was to evaluate the levels of ischemia-modified albumin (IMA) and also other markers of oxidative stress and inflammation in 16 patients with RA and 20 healthy controls. IMA levels were significantly higher in RA patients than healthy controls (0.495 ± 0.01 vs 0.433 ± 0.02 ABSU, P=0.038). No significant differences were observed for the other markers studied. Thus it was concluded that besides RA being related to inflammation, elevated levels of IMA in RA patients suggest that this pathology promotes increased oxidative stress.
A artrite reumatoide (AR) é uma doença inflamatória crônica, de caráter autoimune, caracterizada por poliartrite periférica e simétrica, que leva à deformidade e destruição das articulações devido à erosão da cartilagem e do osso. Esta é uma doença comum que afeta aproximadamente 1% da população mundial, sendo mais frequente em mulheres do que nos homens, no entanto, seu pico de incidência ocorre entre a quarta e sexta décadas de vida. Grandes quantidades de espécies reativas de oxigênio (EROs) foram identificadas no fluido sinovial de pacientes com AR, e essa grande quantidade pode levar a dano oxidativo ao ácido hialurônico, lipídios, matriz da cartilagem e ao DNA. O acumulo de EROs nas células, também serve como importantes moléculas sinalizadoras intracelulares que amplificam a resposta inflamatória - proliferativa sinovial. Assim, o objetivo deste estudo foi avaliar os níveis de albumina modificada pela isquemia (IMA) e outros marcadores de estresse oxidativo e inflamação em 16 pacientes com AR e 20 controles saudáveis. Os níveis de IMA foram significativamente maiores no grupo de pacientes com AR do que os controles saudáveis (0.495 ± 0.01 vs 0.433 ± 0.02 ABSU, P=0.038). Não foram observadas diferenças significativas para os outros marcadores estudados. Desta forma, foi possível concluir que além da AR estar relacionada com a inflamação, os níveis elevados de IMA em pacientes com AR, sugerem que esta patologia promova o aumento do estresse oxidativo.

The etiology of anemia is characterized by abnormal hemoglobin synthesis. Iron deficiency is characterized by microcytic and hipochromic red cells and low serum ferritin, being the most prevalent nutritional deficiency worldwide, responsible for iron deficiency anemia (FA). Anemia of chronic disease (ACD) is considered a clinical syndrome associated with chronic inflammation, infectious disease, neoplastic or traumatic, being the second most frequent cause of anemia. The severity of anemia correlates with the degree of pathology. Both have functional iron deficiency. The objective of this study was to evaluate hematological and inflammatory, as well as the presence of oxidative stress in patients with anemia. The blood analyzer was done by the CBC, automated hematology analyzer processed, Sysmex® (Automated Hematology Analyzer). The quantitative determination of ferritin is serum was done in IMMULITE analyzer. Levels of CRP and AOPP were performed in serum by automated Cobas MIRA® (Roche Diagnostics). Statistical analysis was performed using GraphPad Prism 5. We analyzed 70 patients with microcytic and hypochromic anemia. Of these, 29 (41.43%) were diagnosed as iron deficiency anemia and 41 (58.57%) with anemia of chronic disease. As a control group, we used samples from 44 patients with hematological parameters, serum ferritin, CRP and AOPP normal. The values of MCV, MCH and MCHC significantly lower in iron deficiency anemia. Ferritin levels showed that it can be considered both a measure of iron store as an inflammatory marker. In ACD there is increased production of inflammatory cytokines, which, in turn, increases the concentration of C-reactive protein (CRP). The results indicate that AOPP in both groups with anemia showed increased levels of this marker, which indicates the presence of oxidative stress, probably caused by increased production of free radicals and decreases in enzyme activities of the antioxidant defense system of erythrocytes.
A etiologia das anemias caracteriza-se pela síntese anormal de hemoglobina. A deficiência de ferro é caracterizada por eritrócitos microcíticos e hipocrômicos e por ferritina sérica baixa, sendo a carência nutricional mais prevalente em todo o mundo, responsável pela Anemia Ferropriva (AF). A Anemia de Doença Crônica (ADC) é considerada uma síndrome clínica, associada à inflamação crônica, doença infecciosa, traumática ou neoplásica, sendo a segunda causa mais freqüente de anemia. Ambas apresentam deficiência funcional de ferro. O objetivo deste trabalho foi avaliar parâmetros hematológicos e inflamatórios, bem como a presença de estresse oxidativo em pacientes com anemia. A análise hematológica foi feita através do hemograma, processado em analisador hematológico automatizado, Sysmex® (Automated Hematology Analyzer). O doseamento quantitativo da ferritina no soro foi feito em analisador IMMULITE. A dosagem de Proteína C-Reativa (PCR) e de Produtos da Oxidação Avançada de Proteínas (AOPP) foram realizadas no soro através do sistema automatizado Cobas MIRA® (Roche Diagnostics). A análise estatística foi realizada através do programa GraphPad Prism 5. Foram analisados 70 pacientes portadores de anemia microcítica e hipocrômica. Destes, 29 (41,43%) foram diagnosticados como anemia ferropriva e 41 (58,57%) com anemia de doença crônica. Como grupo controle, foram utilizadas amostras de 44 indivíduos com parâmetros hematológicos, níveis de ferritina, PCR e AOPP dentro da normalidade. Os valores de VCM, HCM e CHCM foram significativamente menores na anemia ferropriva. Os níveis de ferritina revelaram que ela pode ser considerada tanto uma medida das reservas de ferro quanto um marcador inflamatório. Na ADC há aumento da produção de citocinas inflamatórias, que, por sua vez, aumenta também a concentração de PCR. Os resultados do AOPP indicam que ambos os grupos com anemia apresentaram níveis aumentados deste marcador, o que indica a presença de estresse oxidativo, provavelmente causado por aumento na produção de radicais livres e declínio das atividades das enzimas do sistema de defesa antioxidante dos eritrócitos.

INTRODUCTION AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress. RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs. AIM OF THE PROJECT The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify which AGEs/ALEs are produced. Simultaneously, other variables during the sample preparation and analysis were optimized. As explained before, AGEs/ALEs are present in very low concentrations in biological samples, hence the need for very sensitive methods and instrumentation allowing identification. Since human serum albumin (HSA) is the main protein present in plasma, around 50-60%, and has multiple nucleophilic targets, it represents the best model for characterizing AGEs/ALEs. For this reason, the focus was on extracting HSA from plasma, using the newest generation of tribrid MS for the analysis of AGEs/ALEs in plasma samples. AGEs are ligands for RAGE, meaning, they can bind and activate the receptor, inducing a signaling pathway and pro-inflammatory response. ALEs have also been shown to induce a pro-inflammatory response; however, no specific receptor has been linked to this cellular event. Using a cell line with and without RAGE, we aimed to determine whether ALEs can bind and activate the Nf-κB pathway through RAGE. RESULTS AND DISCUSSION ALEs as binder of RAGE In order to investigate the interaction between RAGE and ALEs, different ALEs were produced in-vitro by incubating HSA with different concentrations of well-known lipid derived RCS and in particular: ACR, MDA and HNE. After 24, 48 and 72 h, aliquots of the incubation mixtures were withdrawn, and the reaction was stopped by removing the excess of RCS by ultrafiltration. Intact protein analysis by direct infusion MS was used to evaluate the extent of HSA modifications and demonstrated that by using a wide range of molar ratios and different time-points a quite wide array of ALEs for each tested RCS was generated. In order to characterize ALEs selectively enriched by RAGE, a VC1 pull-down assay was performed as previously described. HSA and HSA treated with MDA, ACR or HNE were assayed for binding to VC1-resins and to control resin. As expected, unmodified HSA was not retained by the VC1-resin. At increasing molar ratios and incubation time, higher amounts of albumin modified with MDA or ACR were eluted from the VC1 resin, with a predominance of the high molecular weight (HMW) species. The modified albumins were retained by the VC1-resin, but not by the control resin. ALEs in the reaction mixtures and those enriched by VC1 were analyzed by bottom-up MS in order to identify the PTMs and to localize the amino acid residues involved in the protein adduct formation. With regard to MDA, only di-hydropyridine adducts on lysines (DHPK), and N-2-pyrimidyl-ornithine adducts on arginines (NPO) were retained by VC1-domain. The n-propenal modifications of lysine (NPK), largely identified before enrichment, were not identified after the enrichment. ACR induced a set of modifications which were identified only after VC1 enrichment and in particular the N-(3-formyl-3,4-dehydro-piperidinyl) lysine (FDPK) modifications, the Michael adduct on cysteines, the double Michael adduct of lysines, the Michael adduct of histidine, the N-2-(4 hydroxy-tetrahydro-pyrimidyl) ornitine (propane-arginine, HTPO) and the Nε-(3-methylpyridinium)-lysine (MP-lysine). Most of the ALEs generated by HNE were found both before or after enrichment, with the exception of a few Michael adducts which were selectively retained by VC1 (not detected before enrichment). With a view to rationalizing the key factors influencing the RAGE binding of the monitored adducts, in silico studies were performed. They were focused on the adducts on arginine and lysine residues as formed by ACR and MDA since they are numerous, with a very broad range of affinity, thus allowing the development of clear structure-affinity relationships. RAGE-ligand interacting regions are characterized by a rich set of positively charged residues which can bind acidic regions of a protein. The mechanism identified using in silico studies, involves a basic amino acid at the center of carboxylic acids like glutamate and aspartate, which forms a set of ionic bridges. Once the basic amino acid is modified by ACR or MDA to an adduct with a neutral charge, the carboxylic acids become available to freely contact the RAGE positive residues. Identification of AGEs/ALEs in biological samples The VC1 technique has proven to be successful in enriching AGEs and ALEs, so the next step was to exploit this technique in biological samples. In order to identify proteins prone to be modified due to oxidative pathways, and possibly serve as biomarker, healthy human plasma was oxidized using the radical initiator AAPH. Different concentrations of AAPH and different timepoints were tested for the presence of protein carbonyl groups, an indicator for protein oxidation and possibly the formation of AGEs/ALEs. A time and concentration dependent formation of carbonyl groups is observed in plasma. Next, samples were analyzed using a bottom-up approach. Results obtained were showing many oxidation products, such as amino side chain oxidation, however no AGEs/ALEs were identified. Thus, a new approach was adopted, including the incubation of plasma directly with RCS, such as HNE, MDA and ACR. This resulted in the formation of AGEs/ALEs in plasma samples, however, they could not be retained by the VC1 domain. Instead of using the VC1 technique to enrich AGEs/ALEs from biological samples, other variables throughout the experimental set-up were optimized. Previously, peptides were analyzed using the Orbitrap LTQ XL, a very powerful instrument. Nonetheless, the newest generation of tribrid MS offers even higher resolution, and it increases protein coverage due to parallel isolation and detection, and faster analyzers. Moreover, we focused on AGEs/ALEs from HSA and using NaBH4 to reduce and stabilize adducts throughout the analysis. This new approach permitted us to identify many AGEs/ALEs in both healthy human plasma samples, but also AGEs/ALEs only present in heart failure samples. Glycation on lysine residues was the main modification identified, present in both healthy and heart failure plasma samples. Important is the HNE Michael adduct, specifically identified in only heart failure samples. Moreover, the importance of stabilizing adducts is underlined by the fact that the acrolein Michael adduct could only be identified after reduction with NaBH4. Development of a cellular assay to determine pro-inflammatory activity of RAGE binders Another part of this project was focused on elucidating whether AGEs/ALEs induce an inflammatory response in cells. For this purpose, a collaboration was started with the Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino. Using a rat epithelial lung cell line overexpressing RAGE, and a control cell line not expressing RAGE, it could be detected if AGEs/ALEs exhibit an effect by binding to RAGE. Moreover, both cell lines were transfected with a Nf-κB reporter gene allowing us a fast and sensitive method for determining if binding of RAGE induces a down-stream signaling pathway. This system implies a firefly luciferase gene downstream from the Nf-κB gene. When the Nf-κB pathway is activated, independently from RAGE, it produces the firefly luciferase enzyme. After adding a luciferin substrate, firefly luciferase is able to convert this substrate into another substrate with light as by-product, which can be measured by a luminometer. IL-1α was used as a positive control, since it induces a strong inflammatory response through Nf-κB. Moreover, known ligands of RAGE able to activate the Nf-κB pathway, were used to validate the cellular experiment, including HSA modified with fructose (AGE), and HMGB1. Results show that Nf-κB is already increased in untreated cells with RAGE and that AGEs induce the Nf-κB pathway independently from RAGE. Moreover, the difference between control and RAGE cells is not significantly increased in the presence of HMGB1 compared to untreated. However, the positive control seemed to induce a much stronger activity in cells with RAGE. Overall, this cellular assay is good for assessing pro-inflammatory activity, however, it is not optimized yet for distinguishing a RAGE-dependent mechanism. CONCLUSION In summary, by using an integrated MS (intact protein and bottom-up approach) and computational approach we have found that some ALEs generated from lipid peroxidation RCS are RAGE binders. We have also found the basic features that ALEs from HNE, MDA and ACR must have to be a RAGE binder: 1) the covalent adducts should greatly reduce or abolish the basicity of the target amino acid, 2) the basic amino acid should be at the center of a set of carboxylic acids which, once the residue is modified, become available to freely contact the RAGE positive residues. Next step was to use the VC1 technique to enrich AGEs/ALEs in biological samples. First, oxidized human plasma was used, however, using the Orbitrap LTQ XL, it was not sufficient to identify AGEs/ALEs. Therefore, analysis was moved to a higher resolution mass spectrometer, which allowed us to identify AGEs/ALEs in plasma samples of heart failure patients, showing the powerfulness of this new generation MS. Important was to understand whether ALEs could induce pro-inflammatory activity through RAGE, since we showed that ALEs are RAGE binders. Unfortunately, the cellular assay that was set up is efficiently in determining Nf-κB dependent pro-inflammatory activity, but not if it is RAGE dependent.

ABSTRACT As far back as the early 1900?s when it was discovered that water could be a mode of transmitting diseases, chlorine was used to disinfect water. In the 1970?s, the formation of disinfection by-products (DBPs) from the reaction of chlorine with natural organic matter was discovered. Since then there have been various studies on alternative disinfectants that could inactivate microorganisms and at the same time form less or no disinfection by-products. More recently the ultraviolet (UV) irradiation has been used to both disinfect and remove organic contaminants in drinking water. Though the use of UV irradiation has been found to be very effective in the inactivation of microorganisms, it does not provide a residual effect to maintain the water?s microbial quality in the distribution system. Due to this, a secondary disinfectant such as chlorine has to be used to achieve microbial stability, suggesting that the formation of chlorination disinfection by-products would still occur but perhaps in different quantities and with different chemical species. In this research, the use of factorial experiments and single factor experiments were used to determine the effects of pH, alkalinity and UV-fluence (dose) on the formation of three classes of disinfection by-products; haloacetic acids (HAAs), haloacetonitriles (HANs) and trihalomethanes (THMs). These disinfection by-products were measured in water samples following post-UV chlorination and the UV treatment was either UV photolysis or UV/H₂O₂. From the factorial experiment results, treatment of synthetic water with UV/H₂O₂, an advanced oxidation process (AOP), produced fewer post-UV chlorination disinfection by-products (PCDBPs) than UV photolysis. For chlorinated PCDBPs, the percentage difference between UV photolysis and UV/H₂O₂ was 55, 65 and 38% for total HAAs (HAA₉), total HANs (THANs) and total THMs (TTHMs) respectively. The percentage difference between UV photolysis and UV/H₂O₂ for brominated PCDBPs was 41 and 42% for HAA9 and TTHMs respectively. Both the use of pH and alkalinity proved to be factors that were significant in affecting the yields of the PCDBPs studied. Increases in alkalinity were found to increase the formation of PCDBPs in the treatment of synthetic water with UV/H₂O₂. Alkalinity had the opposite effect for PCDBP formed under UV photolysis conditions. Increases in pH always decreased the formation of PCDBPs. In the single factor experiments, haloacetic acid concentrations were unaffected as alkalinity was increased but dichloroacetonitrile and chloroform increased in concentration under treatment conditions of UV photolysis followed by chlorination. The UV/H₂O₂ treatment resulted in a decrease in concentration of the PCDBPs. In the pH studies, water samples were subjected only to the UV/H₂O₂ treatments and a reduction in concentration of PCDBPs occurred between pH 7 and 9.

Impaired glucose regulation (IGR) constitutes a spectrum of impaired glucose and metabolic regulation that can result in neuropathy. Several different pathways of injury in the diabetic peripheral nervous system that include metabolic dysregulation induced by metabolic syndrome induce oxidative stress, failure of nitric oxide regulation, and dysfunction of certain key signaling pathways. Oxidative stress can directly injure both dorsal route ganglion neurons and axons. Modulation of the nitric oxide system may have detrimental effects on endothelial function and neuronal survival. Reactive oxidative species can alter mitochondrial function, protein and DNA structure, interfere with signaling pathways, and deplete antioxidant defenses. Advanced glycelation end (AGE) products and formation of ROS are activated by and in turn regulate key signal transduction pathways.

Several measures are used to evaluate kidney function: serum creatinine, urinalysis, renal clearance, and renal imaging. Creatinine is an end product of muscle catabolism and is commonly used as a filtration marker. Dysmorphic erythrocytes in the urinary sediment indicate bleeding in the upper urinary tract. A urine pH less than 5.5 excludes type 1 renal tubular acidosis. A pH greater than 7 suggests infection. Acidic urine is indicative of a high-protein diet, acidosis, and potassium depletion. Alkaline urine is associated with a vegetarian diet, alkalosis and urease-producing bacteria. Clearance of p-aminohippurate is a measure of renal blood flow. Kidney function is evaluated to determine disease states such as glomeruluar disease or tubulointerstitial disease. Clinical manifestations of glomerular injury can vary from the finding of isolated hematuria or proteinuria, or both. In addition, some patients who present with advanced renal insufficiency, hypertension, and shrunken, smooth kidneys are presumed to have chronic glomerulonephritis. Acute and chronic interstitial disease preferentially involves renal tubules.

This chapter discusses the recent advance in meat. Meat is usually a rich source of protein and is also composed of fats, vitamins, and minerals. The composition of these nutrients is different depending on the type of meat. Meat is basically divided into two categories: red and white. Due to high protein and fat content, the chances of oxidation are increased. The oxidation process causes meat spoilage. To protect the meat from spoilage, recent technologies and natural antioxidants are being used. Non-thermal processing techniques including gamma irradiation, e-beam irradiation, high-pressure processing, and pulsed electric field produce safe and quality meat because in recent technologies, conditions can be controlled. Conclusively, recent advances in meat and meat products play a role in improving product life and human health.

Protein glycation is the random, nonenzymatic reaction of sugar and protein induced by diabetes and ageing; this process is quite different from glycosylation mediated by the enzymatic reactions catalysed by glycosyltransferases. Schiff bases form advanced glycation end products (AGEs) via intermediates, such as Amadori compounds. Although these AGEs form various molecular species, only a few of their structures have been determined. AGEs bind to different AGE receptors on the cell membrane and transmit signals to the cell. Signal transduction via the receptor of AGEs produces reactive oxygen species in cells, and oxidative stress is responsible for the onset of diabetic complications. This chapter introduces the molecular mechanisms of disease onset due to oxidative stress, including reactive oxygen species, caused by AGEs generated by protein glycation in a hyperglycaemic environment.

To determine the role of NADPH-oxidase mediated formation of different lipid, protein-derived molecules, and depletion of vitamin-C level in vitreous behind the endothelial dysfunction-induced vascular endothelial growth factor secretion and pathogenesis of diabetic retinopathy (DR) in type 2 diabetes mellitus (T2DM). Fourteen T2DM patients with mild non-proliferative diabetic retinopathy (MNPDR), 11 patients without diabetic retinopathy (DNR), 17 T2 DM subjects with high-risk proliferative diabetic retinopathy (HRPDR), and 5 healthy individuals without DM underwent vitreous analysis for estimation NADPH oxidase, lipid peroxide like malondialdehyde (MDA), 4-Hydroxy-noneal (HNE) and advanced lipoxidation end product (ALE) like Hexanoyl-lysine (HLY), protein carbonyl compound (PCC), Vitamin-C and concentration of vascular endothelial growth factor (VEGF) secretion following standard spectrophotometric methods and enzyme-linked immunosorbent assay (ELISA). Vitreous concentration of NADPH-oxidase, different protein and lipid-derived molecule, and VEGF were found to be significantly elevated among DNR and of DR subjects with different grades compared to HC subjects whereasthe vitamin-C level was found to be decreased among different DR subjects and DNR subjects in comparison to healthy individuals. Oxidative stress-mediated lipid and protein-derived biomolecules not only add important mediators in the pathogenesis of DR, but also accelerate the progression and severity of microangiopathy.

Whole grain wheat has a diverse supply of hydrophilic and lipophilic antioxidants which include phenolic compounds (phenolic acids, flavonoids, anthocyanins which are present only in pigmented wheat, alkylresorcinols, and to a lesser extent proanthocyanidins), carotenoids (mainly lutein and zeaxanthin) and tocochromanols (α, β, γ and δ - tocopherols and tocotrienols). This diversity of antioxidants function to protect consumers from radical-induced oxidative damage caused by various free radicals produced endogenously from metabolic processes and exogenously from sunlight and other chemicals and environmental pollutants. They also chelate metal ions which could catalyze oxidation reactions in the physiological system, thereby providing antioxidant protection. However, phenolic compounds might exert pro-oxidant effect when bound to heavy metal ions. This effect could be prevented by the diverse antioxidant system in wheat.

Diabetic neuropathy (DN) is a serious complication in type-1 diabetes and type-2 diabetes. Animal models show many abnormalities like neuropathy, hyperalgesia, allodynia, slow nerve conduction velocity (NCV), and progressive sensory and motor deficit that are associated with diabetic neuropathy. Various risk factors may be involved in causing DN, such as persistent hyperglycemia, microvascular insufficiency, oxidative stress, nitrosative stress, defective neurotrophism and autoimmune-mediated nerve destruction. Many conventional and newer therapeutic approaches are available. Approaches include effective control of glycemia. Symptoms targeted therapies such as antidepressants, SSRIs, anticonvulsants, opiates, NSAIDs and NMDA receptor antagonists. Therapies targeting particular causes include aldose reductase inhibitors, drugs that act on hexosamine pathways, protein kinase C pathways and AGE receptors. Preclinical studies involving pharmacological agents have shown positive results but were withdrawn at the stage of a clinical study, either due to lack of efficacy or due to their side effects on major organs. Medicinal herbal plants are the richest bio-resource of drugs that have been studied extensively for their neuroprotective effects. Various approaches involving neuroprotection by natural products are discussed here.

With the increasing demand for sustainable and functional proteins from alternative sources, it is necessary to use advanced proteomics and bioinformatics tools for more time and cost-efficient research. The identification and release of abundant proteins/peptides from plant-based sources has been gaining significant attention by the food industry in the last decade. Despite its low protein content (1–2%), the magnitude of proteins obtained from the starch industry (~240,000 tons/year) makes potatoes a highly relevant source as a plant-based protein. Previously, we have identified and validated abundant peptides with good emulsifying and antioxidant properties using bioinformatics and proteomics tools as well as in vitro model systems. Using data-driven targeted hydrolysis, we were able to release validated, functional peptides from the potato protein obtained from potato fruit juice, a protein rich by-product of potato starch production. This work focuses on fractionation of potato protein hydrolysates (PPH) obtained through such targeted hydrolysis using trypsin and subsequent fraction characterization. Unfractionated (PPH1) and membrane-fractionated (PPH2 as >10kDa, PPH3 as 10-5kDa, PPH4 as 5-0.8kDa and PPH5 as < 0.8kDa) PPH was characterized for emulsifying and antioxidant properties/potential. Pendant drop technique and dilatational rheology were applied for determining interfacial tension and viscoelasticity of the PPH fractions at the oil-water interface. PPH2 (>10kDa) showed higher decrease of oil-water interfacial tension. All fractions predominantly provided elastic, weak and easily stretchable interfaces. PPH2 provided more rigid interfacial layer than the other fractions. Radical scavenging and metal chelating activities of PPHs were also tested and the best activities were provided by fractions >5kDa. Furthermore, their ability to form physically and oxidatively stable 5% fish oil-in-water emulsions were investigated during 8-day storage and results generally showed that fractions >5kDa provided the best stability followed by the 5–0.8kDa fraction.

The use of plant proteins to stabilize oil-in-water (O/W) emulsions has been an increasing trend lately. The complexity of the available plant protein ingredients, along with the proteins’ physicochemical properties, require advanced processing that typically leads to substantial concentrations of non-protein components in the final isolates or concentrates. It is known that those components, such as polyphenols, phytic acid or phospholipids, can have a strong influence on the oxidative stability of emulsions. Thus, to understand the oxidative stability of plant protein-stabilized emulsions, the influence of the non-protein components also needs to be considered. Many food emulsions, such as mayonnaise or infant formula, are stabilized by not only proteins, but also phospholipids. Such an interfacial protein-phospholipid combination can also be found in oleosomes, natural lipid droplets which show a high oxidative stability. This stability has been attributed to their interfacial architecture in which oleosins and phospholipids form a tight physical barrier against pro-oxidant species. However, while the antioxidant properties of proteins are widely reported, the contribution of phospholipids to lipid oxidation in plant protein-based emulsions remains underexplored. In this work, we investigated how mixed interfacial plant proteins and phospholipids may be rationally used to control the oxidative stability of O/W emulsions. The interfacial composition was modulated by varying the ratio between pea proteins and sunflower phosphatidylcholine (PC) while keeping the total concentration of pea proteins constant. Increasing the phospholipid-to-protein ratio led to a monotonic decrease in the concentration of proteins and an increase of phospholipids at the interface, while the oxidative stability of those O/W emulsions changed in a non-monotonic pattern. The results were put in perspective by embedding them in a context of reviewing the potential implications of typical components in plant protein ingredients on lipid oxidation.

Chemical Vapor Aluminizing (CVA) is used for more than 20 years to protect blades and vanes in the hot section of aero- and land based turbines against oxidation and hot corrosion [1]. Modern CVA is an advanced process capable of controlled alloying the coating with additional elements using metal chlorides and tight control of the coating composition. CVA processes offer a number of advantages over conventional pack and above-the-pack cementation. This paper deals with the industrial CVA technology to produce multi-component coatings using different metal chloride generating devices. Specific examples illustrate the influence of the coating parameters and base materials on the NiAl-based coatings microstructure and composition. Advanced co-deposition CVA processes with addition of different metal elements to the aluminide coatings are presented. Modified coating properties and structures of multiple metal coatings with elements like Al, Cr, Si, Co, Y and others will be discussed.

Mechanically expelled soy cake (MESC) is a valuable source of protein and other nutrients; however, this product stream is underutilized for human consumption, especially in low-resource settings such as the Sub-Saharan Africa (SSA) region. Additionally, MESC still contains oils, which are prone to oxidation. Through collaborative partnerships with scientists like Dr. Keshun Liu, USDA, ARS, and Dr. Kephas Nowakunda, NARL, we piloted a low resource method to make soy protein concentrate (SPC) that can be widely used by processors in developing economies. Using this method, oil content, oxidized byproducts, phytic acid, and oligosaccharides are reduced, while increasing protein content to almost 63% db. In this session, we demonstrate an alternative low-resource method to produce SPC from MESC and evaluate their nutritional quality and oxidative stability. We also present early sensory findings and hypothesize on potential uses and benefits of SPC products and ingredients in emerging markets. The SPC materials produced were characterized based on their protein, oil and phytic acid contents. Thiobarbituric acid reactive species using malondialdehyde (MDA) as standard and peroxide value were assessed in SPC meal and oil extracted from SPC, respectively, to evaluate oxidation status in comparison with raw materials.With this alternative less resource-demanding method, SPC with a protein content of ~63% db and with lower oil (50% less) and phytic acid (70% less) contents and higher stability due to lower peroxide value (50% less) and MDA (50% less) contents than the original MESC can be made from defatted or low-fat soy flour using locally-available materials and equipment. This is beneficial because with this alternative method, the utilization of SPC for human consumption in SSA can be improved by expanding its incorporation into various food products including extruded snacks, composite flours, porridges, and bakery products.

Preventing lipid oxidation, especially at the oil-water interface, of emulsions is still a challenge issue for suppressing food deterioration. A promising alternative is to introduce polyphenols together with amphiphilic proteins to form protein-polyphenol conjugates. Protein-polyphenols conjugates prepared via chemical bonding possess advantages of enhanced product stability, better controlling/turnability, more stable emulsion. Recently, we have successfully conjugated a natural antioxidant, gentisic acid (GA), into a great protein emulsifier, β-lactoglobulin (βLG) and discovered the significantly enhanced antioxidant activity in an in vitro assay. Moreover, we also proved the possibility of controlling GA grafting number for optimizing antioxidant activity of conjugates. In this follow-up study, we show a strategy to prepare future food ingredients with dual functions of emulsification and antioxidant activity for food emulsions formation. In particular, we systematically investigated the feed ratio of GA and βLG on the structural changes of the protein, as well as the long-term physical stability and oxidative stability of hemp oil-in-water emulsions. Upon optimizing the conjugate production at a larger scale and a thorough characterization of the surface charge, surface hydrophobicity, and surface tension of the resultant conjugates, we found that the feed ratio is extremely important because it determines the solubility, structure and surface activities of conjugated protein and GA grafting number, further produce a great effect on the physicochemical stabilities of their stabilized emulsions. In addition, the emulsions stabilized by the soluble portions of the conjugates could maintain the good physical stability while greatly enhancing the oxidative stability. We also proposed a possible explanation of the correlation between physicochemical stability of emulsions and GA grafting number. This work represents the structural basis of the functional improvement of the emulsions using protein-phenolic conjugates, which provides unique insights into the future design of multifunctional food biopolymers.

Several technologies exist or are under development for treating spent oxide nuclear fuels. Foremost among these are aqueous and pyrochemical reprocessing which both involve a head-end fuel dissolution step. This dissolution step may potentially be shortened if it is combined with a fuel decladding and size reduction process. Declad and Oxidize (DEOX), an advanced head-end processing concept, is being assessed at Argonne National Laboratory to meet these decladding and size reduction needs via the oxidation of UO2 to U3O8. This work is being done in collaboration with Oak Ridge National Laboratory. The primary objectives of the DEOX process are to generate suitable feed material for these two fuel treatment processes and to collect information about the behavior of spent fuel during DEOX processing. Specifically, DEOX is intended to remove the spent fuel from its cladding, while avoiding oxidation of the cladding that would contaminate the product. An additional goal is to obtain a product particle size distribution between 45μm to 4mm. Data will be collected on the extent of fuel oxidation and on the volatilization of fission products. The experimental apparatus used to perform these experiments is described in this report along with preliminary test results.

Aldehydes are major intermediates in oxidation and pyrolysis of hydrocarbons and particularly biofuels. While the high temperature oxidation chemistry of C3-C5 aldehydes have been studied in the literature, a comprehensive low temperature kinetics remains unaddressed. In this work, acetaldehyde, propanal, and 2-propenal (acrolein) oxidation was investigated at low-temperature combustion condition (500–700 K). The isomer specific products concentrations as well as the time-resolved profiles were studied using Sandia’s multiplexed photoionization mass spectroscopy (MPIMS) with synchrotron radiation from the Advanced Light Source (ALS). The laser pulsed photolysis generates chlorine atoms which react with aldehydes to form the parent radicals. In the presence of excess oxygen, these radicals react with O2 and form RO2 radicals. The temperature dependent products yields are determined for 500 K to 700 K and the competition between the channels contributing to the formation of each product is discussed. In acetaldehyde oxidation, the formation of the main products are associated with HO2 elimination channel from QOOH or direct H atom elimination from the parent radicals. In propanal oxidation, the most intensive signal peak was associated with acetaldehyde (m/z=44) which was formed through the reaction of α′-R with O2. α′-RO2 intermediate decomposes to acetaldehyde+OH+CO via Waddington mechanism and formation of five-member ring transition state. In 2-propenal oxidation, the unsaturated radical produced from α-R reacts with O2 to form the primary products.

Canfor, a producer of lumber, pulp and paper needed a solution to replace aging 30-inch (760mm) fiberglass reinforced pipe. A new PE-RT product now expands PE into industrial applications requiring resistance to high temperatures and having a Hydrostatic Design Basis (HDB) of 800psi (55 bar) at 180°F (82.2°C). Through chemical processes, Canfor cooks, washes, and extracts pulp fiber from wood that results in both acidic and caustic effluent with temperatures normally in the 50–60°C range or as high as 70–75°C. Traditional fiberglass pipes have experienced repeated joint failures over time, whereas heat-fused HDPE pipe provides solutions reducing unnecessary maintenance and a longer service life. Standard PE4710 High Density Polyethylene Pipes (HDPE) have pressure ratings limited to 140°F (60°C) and are not normally acceptable for such high temperature acidic and caustic effluent. Additionally, the potential for higher oxidation-reduction potential (ORP) from residual chlorine levels and bleaching also justified turning to a different material based on the potential oxidative attack at high temperatures. The new PE-RT resin protects against oxidative attacks at high temperatures and the flexible heat-fused HDPE pipe provides considerable cost savings during installation. Compared to fiberglass, up to eight 40-foot lengths of HDPE pipe can be joined by heat fusion per day, whereas only two 6-meter (20-foot) lengths of FRP pipe can be wrapped per day. The presentation will highlight photos during the installation process and report the advantages of using the new pipe material. This project provides reference for expanding HDPE pipe into new applications using PE-RT materials.

The timing of dormancy induction and release is very important to the economic production of table grape. Advances in manipulation of dormancy induction and dormancy release are dependent on the establishment of a comprehensive understanding of biological mechanisms involved in bud dormancy. To gain insight into these mechanisms we initiated the research that had two main objectives: A. Analyzing the expression profiles of large subsets of genes, following controlled dormancy induction and dormancy release, and assessing the role of known metabolic pathways, known regulatory genes and novel sequences involved in these processes B. Comparing expression profiles following the perception of various artificial as well as natural signals known to induce dormancy release, and searching for gene showing similar expression patterns, as candidates for further study of pathways having potential to play a central role in dormancy release. We first created targeted EST collections from V. vinifera and V. riparia mature buds. Clones were randomly selected from cDNA libraries prepared following controlled dormancy release and controlled dormancy induction and from respective controls. The entire collection (7920 vinifera and 1194 riparia clones) was sequenced and subjected to bioinformatics analysis, including clustering, annotations and GO classifications. PCR products from the entire collection were used for printing of cDNA microarrays. Bud tissue in general, and the dormant bud in particular, are under-represented within the grape EST database. Accordingly, 59% of the our vinifera EST collection, composed of 5516 unigenes, are not included within the current Vitis TIGR collection and about 22% of these transcripts bear no resemblance to any known plant transcript, corroborating the current need for our targeted EST collection and the bud specific cDNA array. Analysis of the V. riparia sequences yielded 814 unigenes, of which 140 are unique (keilin et al., manuscript, Appendix B). Results from computational expression profiling of the vinifera collection suggest that oxidative stress, calcium signaling, intracellular vesicle trafficking and anaerobic mode of carbohydrate metabolism play a role in the regulation and execution of grape-bud dormancy release. A comprehensive analysis confirmed the induction of transcription from several calcium–signaling related genes following HC treatment, and detected an inhibiting effect of calcium channel blocker and calcium chelator on HC-induced and chilling-induced bud break. It also detected the existence of HC-induced and calcium dependent protein phosphorylation activity. These data suggest, for the first time, that calcium signaling is involved in the mechanism of dormancy release (Pang et al., in preparation). We compared the effects of heat shock (HS) to those detected in buds following HC application and found that HS lead to earlier and higher bud break. We also demonstrated similar temporary reduction in catalase expression and temporary induction of ascorbate peroxidase, glutathione reductase, thioredoxin and glutathione S transferase expression following both treatments. These findings further support the assumption that temporary oxidative stress is part of the mechanism leading to bud break. The temporary induction of sucrose syntase, pyruvate decarboxylase and alcohol dehydrogenase indicate that temporary respiratory stress is developed and suggest that mitochondrial function may be of central importance for that mechanism. These finding, suggesting triggering of identical mechanisms by HS and HC, justified the comparison of expression profiles of HC and HS treated buds, as a tool for the identification of pathways with a central role in dormancy release (Halaly et al., in preparation). RNA samples from buds treated with HS, HC and water were hybridized with the cDNA arrays in an interconnected loop design. Differentially expressed genes from the were selected using R-language package from Bioconductor project called LIMMA and clones showing a significant change following both HS and HC treatments, compared to control, were selected for further analysis. A total of 1541 clones show significant induction, of which 37% have no hit or unknown function and the rest represent 661 genes with identified function. Similarly, out of 1452 clones showing significant reduction, only 53% of the clones have identified function and they represent 573 genes. The 661 induced genes are involved in 445 different molecular functions. About 90% of those functions were classified to 20 categories based on careful survey of the literature. Among other things, it appears that carbohydrate metabolism and mitochondrial function may be of central importance in the mechanism of dormancy release and studies in this direction are ongoing. Analysis of the reduced function is ongoing (Appendix A). A second set of hybridizations was carried out with RNA samples from buds exposed to short photoperiod, leading to induction of bud dormancy, and long photoperiod treatment, as control. Analysis indicated that 42 genes were significant difference between LD and SD and 11 of these were unique.

Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.

This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.