bluuebunny/crossref_metadata_2025 huggingface dataset:
Flagellin enhances NK cell proliferation and activation directly and through dendritic cell-NK cell interactions
Authors: Tsujimoto Hironori , Uchida Takefumi , Efron Philip A , Scumpia Philip O , Verma Amrisha , Matsumoto Tadashi , Tschoeke Sven K , Ungaro Ricardo F , Ono Satoshi , Seki Shuhji , Clare-Salzler Michael J , Baker Henry V , Mochizuki Hidetaka , Ramphal Reuben , Moldawer Lyle L
Published: July 2005

Flagellin, the principal component of bacterial flagella, is a ligand for Toll-like receptor 5 (TLR5) or TLR11 and contributes to systemic inflammation during sepsis through activation of dendritic cells (DCs) and other cells of the innate immune system. Here, we report that flagellin and the TLR4 ligand, lipopolysaccharide (LPS), induced phenotypic and functional maturation of murine bone marrow-derived DCs and enhanced DC accumulation in the draining popliteal lymph node following their footpad injection. It is interesting that flagellin injection enhanced myeloid (CD8α−1) and plasmacytoid (plasmacytoid DC antigen+ B220+) DC subsets, whereas LPS only increased myeloid DCs in the draining lymph node. In addition, the footpad injection of flagellin or LPS induced significant CD4+ T cell activation in the draining popliteal lymph node, as judged by increased CD69 or CD25 expression. We illustrate, for the first time, that flagellin also increases natural killer (NK) cell number and activation status in the draining lymph node after footpad injection. Using coculture with enriched carboxy-fluorescein diacetate succinimidyl ester-labeled NK cells, flagellin-treated DCs induce significant NK cell proliferation and activation. In fact, direct treatment of NK cells with flagellin induces a greater increase in cell proliferation than treatment with LPS. In contrast, flagellin treatment of NK cells was not a strong inducer of interferon-γ (IFN-γ) production, indicating that NK cell proliferation and IFN-γ production may be regulated differentially. These data suggest that flagellin is a capable maturation agent for murine myeloid-derived DCs, and flagellin-activated DCs and flagellin itself are potent inducers of NK cell proliferation.
The kringle domain of urokinase-type plasminogen activator potentiates LPS-induced neutrophil activation through interaction with αVβ3 integrins
Authors: Kwak Sang-Hyun , Mitra Sanchayita , Bdeir Khalil , Strassheim Derek , Park Jong Sung , Kim Jael Yeol , Idell Steven , Cines Douglas , Abraham Edward
Published: July 2005

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. In addition, uPA has been shown to have proinflammatory properties, particularly in potentiating lipopolysaccharide (LPS)-induced neutrophil responses. To explore the mechanisms by which uPA exerts these effects, we examined the ability of specific uPA domains to increase cytokine expression in murine and human neutrophils stimulated with LPS. Whereas the addition of intact uPA to neutrophils cultured with LPS increased mRNA and protein levels of interleukin-1β, macrophage-inflammatory protein-2, and tumor necrosis factor α, deletion of the kringle domain (KD) from uPA resulted in loss of these potentiating effects. Addition of purified uPA KD to LPS-stimulated neutrophils increased cytokine expression to a degree comparable with that produced by single-chain uPA. Inclusion of the arginine-glycine-aspartic but not the arginine-glycine-glutamic peptide to neutrophil cultures blocked uPA kringle-induced potentiation of proinflammatory responses, demonstrating that interactions between the KD and integrins were involved. Antibodies to αV or β3 integrins or to the combination of αVβ3 prevented uPA kringle-induced enhancement of expression of proinflammatory cytokines and also of adhesion of neutrophils to the uPA KD. These results demonstrate that the KD of uPA, through interaction with αVβ3 integrins, potentiates neutrophil activation.
Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells
Authors: Titos Esther , Clària Joan , Planagumà Anna , López-Parra Marta , González-Périz Ana , Gaya Joan , Miquel Rosa , Arroyo Vicente , Rodés Joan
Published: July 2005

Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl4)-induced damage. Rats treated with CCl4 showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl4 treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B4 and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A4, an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.
Examination of urinary mercury levels in dentists in Turkey
Authors: Karahalil B , Rahravi H , Ertas N
Published: July 2005

Mercury (Hg) is a naturally occurring element and its toxicity, especially in certain forms, has been known for many years. Exposure to Hg can occur in occupational and environmental settings. The toxicity of Hg compounds in dentistry has been an issue of increasing concern. Dental personnel are occupationally exposed to Hg vapor in their working environment and this exposure constitutes a potential risk to people in the dental surgery, mainly from the inhalation of Hg vapor and fine particles of amalgam. In this study, the urinary Hg excretion levels of 20 dentists and nine control subjects, matched for age, were determined by cold-vapor atomic absorption spectrometer (CV-AAS). The levels of Hg in the urine samples of the dentists was about three times higher than the control subjects (6.29±3.5 and 1.979±0.9 μg/L, respectively) (P B < 0.001). Some 90% of dentists wore both gloves and masks. Standards of hygiene (use of mask, glove and gown) may contribute to the degree of exposure. Attention to important hygiene measures, such as the avoidance of spills of Hg, cleaning of floors after such spills, ventilation and the installation of ventilation, depending on technology, should be taken into consideration.

Age and smoking habits did not influence the urinary Hg excretion. Our results showed that dentists had significant exposure to Hg vapor compared to control subjects and therefore might be subject to possible adverse effects due to Hg toxicity.
Evidence that iron overload plus croton oil induce skin tumours in mice
Authors: Rezazadeh H , Nayebi A R , Garjani A , Sheikhulislami A , Babaei H
Published: July 2005

Iron overload is known to occur due to different factors including genetic disorders. It has been shown that iron overload predisposes humans to an increased risk of cancer. However, the mechanism by which iron overload enhances chemically induced carcinogenesis is not known. In this report, for the first time it is shown that iron overload acts as a tumour initiator. Female albino Swiss mice were given iron dextran 1 mg/mouse per day intramuscularly for 15 days and croton oil 0.5 mg in 200 mL acetone/mouse topically twice a week for 30 weeks. During this period, the animals were observed for tumour incidence. There were significantly higher yields of tumours in those animals receiving both iron and croton oil. However, the group of animals treated only with acetone, iron, croton oil and 7,12-dimethylben-z(a)anthracene (DMBA) alone did not develop any tumours during 30 weeks of observation. Further, croton oil-mediated induction in cutaneous lipid peroxidation (LPO) level was higher in the iron-overload group. The results of this study suggest that oxidative stress generated by iron overload is responsible for croton oilmediated skin carcinogenesis.
Developmental exposure to fenproporex: reproductive and morphological evaluation
Authors: Moreira C Q , Faria M JSS , Baroneza J E , Oliveira R J , Moreira E G
Published: July 2005

This study was designed to evaluate the maternal toxicity and teratogenicity of fenproporex, one of the most widelyused anorectic drugs in many countries, including Brazil. Three periods of exposure were evaluated: (a) 30 days before mating; (b) from gestational day (GD) 0 to 14; and (c) 30 days before mating and during pregnancy, until GD 14. Female mice from experimental groups received, by gavage, 15 mg/kg of fenproporex. Treatment with fenproporex increased ambulation of dams in the open field test and did not influence the mobility in the forcedswimming test. There was no significant difference in maternal weight gain between the controls and fenproporex-treated groups, although fenproporex treatment reduced the gravid uterus weight. No significant difference was observed in postimplantation loss, fetal viability and sex ratio. In addition, this compound did not impair intra-uterine growth. The reduction in the number of implantations in the groups receiving fenproporex indicates that this drug may have an adverse effect on implantation. Fenproporex treatment also increased the number of fetuses presenting small kidneys and cervical ribs. The present results indicate that fenproporex, in the dose and exposure periods tested, appears to exhibit a low maternal toxicity and teratogenic potential in mice.
Behavioral neurotoxicity in adolescent and adult mice exposed to fenproporex during pregnancy
Authors: Moreira C Q , Faria M JSS , Moreira E G
Published: July 2005

We investigated the effects of gestational exposure to fenproporex, one of the most used anorectic drugs in Brazil, on the behavior of adolescent and adult pups (30 and 60 days of age, respectively). Pregnant Swiss mice were treated daily, by gavage, with 15 mg/kg of fenproporex chloride or water during the whole gestational period. Male pups were submitted to open-field, forced swimming test, tail suspension test and fenproporexinduced stereotyped behavior. The results demonstrated that gestational exposure to fenproporex induces antidepressant-like effect and decreases fenproporexinduced stereotyped behavior in both adolescent and adult pups. Moreover, fenproporex-exposed adolescent pups tended (P–0.06) to be more active than control pups. Our data show, for the first time, that gestational exposure to fenproporex leads to long-lasting behavioral toxicity in male mice characteristic of altered dopaminergic transmission.
Folic acid calls to poison centers in Texas, 1998–2003
Authors: Forrester M B
Published: July 2005

Folic acid (folacin, pteroylglutamic acid) is a mono-glutamate form of the water-soluble B vitamin that is involved in the synthesis of nucleotides and amino acids and the normal maturation of red blood cells. This study describes the folic acid calls received by Texas poison centers during 1998–2003. There were 650 calls involving folic acid as a single-ingredient product, of which 55.1% were human exposures. Children age B / 6 years accounted for 80.1% of the human exposures. Patients were managed outside of the health care facilities in 92.1% of the cases. Of those cases with a known medical outcome, 94.8% had no clinical effects. This study found folic acid exposures reported to poison centers were unlikely to have more than minor adverse affects.
Historical Perspective of the Maillard Reaction in Food Science
Authors: FINOT PAUL‐ANDRÉ
Published: July 2005

Abstract:Maillard's paper of 1912 describing the reaction between amino acids and sugars is both innovative and visionary. It provides original and still‐valuable data on the chemistry of a new reaction and foresees its involvement in many scientific and biological domains, even in human pathology. This paper was ignored by the scientific community until 1941. In 1948 the Maillard reaction was definitely recognized as being responsible for the browning and loss of nutritive value of heated milk powders. There was then a continuous increase in papers on the chemistry of this complex reaction to identify its various pathways: in food science, to evaluate the influence of reaction parameters (pH, T°, time, sugar reactivity, concentration of the reagents, water activity, glass transition temperature) on the evolution of the reaction and on changes in food quality; in nutrition, to quantify the loss of bioavailability of essential amino acids; on the metabolism of the reaction products and on the physiological effects of the ingested Maillard reaction products. The significant scientific advances and the key persons and pioneers who contributed much to the understanding of the Maillard reaction are presented. The food industry is directly concerned with the occurrence of this reaction in processed foods and contributed significantly by its own research to understanding the phenomena and to optimizing the processes and conditions of food preparation in order to preserve the nutritional, safety, and organoleptic qualities of foods.
The Discovery of Glycated Hemoglobin: A Major Event in the Study of Nonenzymatic Chemistry in Biological Systems
Authors: RAHBAR SAMUEL
Published: July 2005

Abstract: Glycated hemoglobins are minor components of human hemoglobin (Hb). These are formed nonenzymatically by condensation of glucose or other reducing sugars with α‐ and β‐chains of hemoglobin A. The subfraction HbA1c, a nonenzymatic glycation at the amino‐terminal valines of the β‐chain, was identified by the author in the 1960s as a minor “abnormal fast‐moving hemoglobin band” in diabetic patients during routine screening for hemoglobin variants. This finding later turned out to be an important biomolecular marker with clinical and pathological applications. Measurement of HbA1c in diabetic patients is an established procedure for evaluating long‐term control of diabetes, and the introduction of this measurement represents an outstanding contribution to the quality of care of diabetic patients in this century. More importantly, HbA1c is the first example of in vivo nonenzymatic glycation of proteins, and its discovery opened new and still‐growing avenues of research on Maillard reactions in biological systems, including the concept of advanced glycation/lipoxidation end products (AGEs/ALEs) and the development of diabetic complications and various diseases associated with aging. Although interest in the Maillard reaction is growing rapidly, much remains to be done in this field, including detection and characterization of all in vivo AGEs/ALEs, development and clinical applications of AGE inhibitors and breakers, as well as investigations into the possible roles of the Maillard reaction in regulatory biology and carcinogenesis.
Taste‐Active Maillard Reaction Products: The “Tasty” World of Nonvolatile Maillard Reaction Products
Authors: HOFMANN THOMAS
Published: July 2005

Abstract: This study was done to obtain greater insight into the structures and sensory activities of those tastants that are not present in foods per se, but are generated during food processing by Maillard‐type reactions from carbohydrates and amino acids and thus remain unknown. In order to rank the tastants according to their relative taste impact and to identify the key tastants generated during thermal food processing, the so‐called taste dilution analysis (TDA), which uses the human tongue as a biosensor for tastants, was applied to heated, intensely bitter tasting binary mixtures of glucose or xylose and proline or alanine, respectively. This screening technique led to the identification of previously unknown taste compounds, among which intensely bitter tastants such as quinizolate and homoquinizolate, a pungent‐tasting pyranopyranone, cyclopentenone derivatives exhibiting a physiological cooling effect, as well as a taste‐enhancing pyridinium betaine named alapyridaine will be presented.
Oxidative Pyrolysis and Postpyrolytic Derivatization Techniques for the Total Analysis of Maillard Model Systems: Investigation of Control Parameters of Maillard Reaction Pathways
Authors: YAYLAYAN V.A. , HAFFENDEN L , CHU F L , WNOROWSKI A
Published: July 2005

Abstract: Factors that regulate various pathways of Maillard reaction leading to aroma, color, or carcinogen generation have not been identified, due to the difficulties associated with analyzing complex reaction mixtures. In particular, the role played by oxidation in directing aromagenic, chromogenic, or carcinogenic pathways is not well understood. In order to overcome the analytical difficulties, novel Py‐GC/MS‐based methodologies were developed to analyze volatile and nonvolatile residues of Maillard reaction products generated from the same model system under air or helium atmosphere. The analysis of nonvolatiles was achieved through a postpyrolytic in situ derivatization technique using hexamethyldisilazane, and pyrolysis under air was achieved through modification of the GC equipped with sample concentration trap to allow gas stream switching and subsequent isolation of the pyrolysis chamber from the analytical stream. In this approach label incorporation from the starting materials can be observed in both volatile and nonoxidative conditions for mechanistic studies. In addition, monitoring of redox potentials, oxygen consumption, and color generation of relevant model systems over time were also carried out at different temperatures. The data collected have indicated that perturbation in the redox potential of Maillard model systems by external (oxidizing conditions) or internal (formation of reductones) factors can alter the balance among the four critically important groups of precursors: α‐dicarbonyl, α‐hydroxycarbonyl, 2‐aminocarbonyls, and 2‐(amino acid)‐carbonyl compounds and hence control the relative importance of aromagenic versus chromogenic pathways.
Identification of N7‐(1‐Carboxyethyl)‐Arginine, a Novel Posttranslational Protein Modification of Arginine Formed at High Hydrostatic Pressure
Authors: ALT NADJA , SCHIEBERLE PETER
Published: July 2005

Abstract: An aqueous solution of Nα‐acetylarginine (0.1 mol/L) and glucose (0.3 mol/L) in 0.5 mol/L 3‐[N‐morpholino]propanesulfonic acid (5 mL; pH 7.0) was incubated at 110°C for 2 h with application of high hydrostatic pressure (HHP). After enzymic deacetylation, the arginine modifications formed were separated by ion exchange chromatography. Besides N5‐(4,5‐dihydro‐4‐methyl‐5‐oxo‐1H‐imidazol‐2‐yl)‐L‐ornithine, the novel arginine modification N7‐(1‐carboxyethyl)‐arginine was identified based on NMR and LC/MS measurements and synthesis—in particular, in samples reacted at a pressure of 600 MPa. As compared to samples treated at normal pressure, both compounds were formed in higher amounts at increased pressure.
Labeling Studies on the Formation Pathway of Nε‐Carboxymethyllysine in Maillard‐type Reactions
Authors: KASPER MARTINA , SCHIEBERLE PETER
Published: July 2005

Abstract: Nε‐carboxymethyllysine (CML) is among the most often studied advanced glycation end products (AGEs) in foods and medicine, and several pathways have been suggested for its formation. Based on the results of model studies using glucose labeled with carbon‐13 in different positions, a new pathway is proposed for CML formation without involving free glyoxal or autoxidation reactions.
Elucidation of Chemical Pathways in the Maillard Reaction by 17O‐NMR Spectroscopy
Authors: ROBERT FABIEN , VERA FRANCIA ARCE , KERVELLA FRANK , DAVIDEK TOMAS , BLANK IMRE
Published: July 2005

Abstract: 17O‐NMR spectroscopy was employed as an innovative method to help understand mechanistic pathways in sugar fragmentation. Elucidation of reaction mechanisms to final Maillard end products was achieved by starting from specific intermediates obtained by synthesis, such as 1‐deoxy‐d‐erythro‐hexo‐2,3‐diulose. This α‐dicabonyl was thermally treated in the presence of 17O‐enriched water under alkaline conditions. The reaction products were monitored by 17O‐NMR spectroscopy and their structures corroborated by complementary techniques. For the first time, evidence is shown for the direct formation of acetic acid from 1‐deoxy‐d‐erythro‐hexo‐2,3‐diulose by an oxidative α‐dicarbonyl cleavage and incorporation of a 17OH group into the acetic acid released as sugar fragment.
The Effect of Reaction Conditions on the Origin and Yields of Acetic Acid Generated by the Maillard Reaction
Authors: DAVIDEK TOMAS , DEVAUD STÉPHANIE , ROBERT FABIEN , BLANK IMRE
Published: July 2005

Abstract: The effect of the reaction conditions on the origin and yields of acetic acid from glucose was studied in the system containing equimolar concentrations of 13C‐labeled glucose and glycine. Acetic acid was quantified by GC‐MS using isotope dilution assay. The β‐dicarbonyl cleavage of 1‐deoxyhexo‐2,4‐diulose is proposed to be a major pathway leading to the formation of acetic acid in the glucose‐based Maillard reaction systems under food processing conditions. Acetic acid was built up from all six carbon atoms of glucose. The relative distribution of acetic acid was independent of the reaction time. Temperature and pH had only small effects.
Formation of Mutagenic/Carcinogenic Heterocyclic Amines under Moderate Conditions
Authors: KINAE NAOHIDE , MORI CHISATO , KUJIRAI KAORI , MASUMORI SHOJI , MASUDA SHUICHI
Published: July 2005

Abstract: More than 10 kinds of heterocyclic amines (HCAs), showing mutagenic and carcinogenic potency, have been isolated from cooked fish and meat. But many researchers say that the contribution ratio of HCAs to human cancer is very low. Our purpose in this experiment was to investigate the possibility of the formation of HCAs under moderate conditions, including in vivo. A mixture of d‐glucose, creatinine, and amino acid such as glycine, methionine, threonine, and proline was dissolved in phosphate‐buffered solution (pH7.4) and incubated at 37°C, 50°C, 128°C. At an appropriate time, an aliquot of the reaction solution was treated with blue cotton. HCAs were separated from the blue cotton by elution with 2% ammoniacal methanol. The eluates were submitted to the Ames test, the micronucleus test for determination of mutagenicity, and also LC‐MS analysis for the detection of HCAs. Nonadsorbates to blue cotton were treated with dichloromethane and then subjected to the mutagenicity test. In the Ames test, the mutagenic activity of the reaction mixture increased with an increase of the reaction temperature. The HCA fraction from 50°C incubated solution showed high frequency in the micronucleus test using HepG2 cells. The dichloromethane fractions contained other type of mutagens different from HCAs. In HCA fractions, IQ, MeIQx, 4,8‐DiMeIQx, and 7,8‐DiMeIQ were identified. It is said that the heating process is an essential factor in the formation of HCAs. But our experiment shows that HCAs are produced not only in the cooking process, but also in moderate conditions such as 37°C and 50°C.
Maillard Reactions of Ribose 5‐Phosphate and Amino Acids
Authors: SANDWICK ROGER , JOHANSON MATTHEW , BREUER ELIZABETH
Published: July 2005

Abstract: An important metabolite in nucleotide synthesis, ribose 5‐phosphate (R5P) undergoes Maillard reactions at a rate significantly faster than most common sugars and sugar phosphates of its type. At pH = 8 and 37°C, R5P reacts with amino acids to generate UV‐absorbing compounds within hours and brownish compounds within a day. The increased rate can be attributed to the strategic location of the 5‐phosphate group near the reactive C1 center. As expected, the rate of reaction is at least partially related to pKa of the attacking amine and it follows first‐order kinetics in respect to amine (in the case of glycine). In terms of producing 280 nm absorbing compounds, the reaction order in respect to R5P was 1.5. The reaction mechanism appears to proceed through a 1‐deoxyribosone intermediate. In reactions with proteins, R5P exhibits an ability to promote cross‐linking.
Protein Cross‐Linking in Food
Authors: GERRARD J.A. , MEADE S J , MILLER A G , BROWN P K , YASIR S B M , SUTTON K H , NEWBERRY M P
Published: July 2005

Abstract: The aims of this paper are (1) to probe the relationship between molecular structure and protein cross‐linking ability for a range of small molecules; (2) to establish whether this relationship holds within a food matrix; and (3) to test the impact of Maillard cross‐linking on food functionality, particularly texture, in wheat‐ and soy‐based food systems. A variety of molecules were obtained, either commercially or via organic synthesis. Cross‐linking ability was tested using our standard model system, employing ribonuclease A and analyzing the results by SDS‐PAGE. Molecules of varying reactivity were tested in wheat‐ and soy‐based products, and the changes in functionality were correlated with changes in protein cross‐linking. No simple relationship was found between molecular structure and ability to cross‐link ribonuclease. Only the most reactive reagents were able to cross‐link within the food matrix. Nevertheless, a low degree of cross‐linking was shown to have significant consequences on the properties of wheat‐ and soy‐based foods, suggesting that the Maillard reaction may represent a means to control food texture.
Chemistry and Biological Effects of Melanoidins and Glyceraldehyde‐Derived Pyridinium as Advanced Glycation End Products
Authors: HAYASE FUMITAKA , USUI TERUYUKI , NISHIYAMA KAZUYUKI , SASAKI SHIGEYUKI , SHIRAHASHI YOSHINOBU , TSUCHIYA NAOYUKI , NUMATA NAOKO , WATANABE HIROHITO
Published: July 2005

Abstract: Blue pigments (blue‐M1 and blue‐M2) and red pigments (red‐M1 and red‐M2) were generated in a xylose‐glycine reaction system. Blue‐M2 was identified as an addition compound of di‐xylulose‐glycine to blue‐M1 that involved two pyrrolopyrrole structures. We identified red pigments as isomers of addition compounds of xylulose‐glycine to the condensed compound between pyrrolopyrrole‐2‐carbaldehyde and pyrrole‐2‐carbaldehyde. These pigments have polymerizing activity, suggesting that they are important Maillard reaction intermediates through the formation of melanoidins. Melanoidins induced IFN‐γ and IL‐12 expression in spleen cells exposed to allergen and in macrophages, respectively. These findings suggest that melanoidins have a suppressive effect on allergic reaction as a novel physiological effect. On the other hand, we identified a glyceraldehyde‐derived advanced glycation end product (AGE) formed from glyceraldehyde and N‐acetylarginine as well as glyceraldehyde‐derived pyridinium (GLAP) in physiological conditions. The AGE was identified as 5‐methylimidazoline‐4‐one (MG‐H1), which has been reported to be formed from arginine and methylglyoxal. GLAP, which induced reactive oxygen species (ROS) production in HL‐60 cells, is supposed to be a toxic AGE, while MG‐H1 is a nontoxic AGE.
Ornithine Is a Novel Amino Acid and a Marker of Arginine Damage by Oxoaldehydes in Senescent Proteins
Authors: SELL DAVID R. , MONNIER VINCENT M.
Published: July 2005

Abstract: Long‐lived proteins undergo age‐related postsynthetic modifications by glycation and advanced glycation end products (AGEs), which destabilize them by altering their conformation and charge. It was accidentally discovered that ornithine (orn) increased with age in acid hydrolyzates of human skin collagen and lens crystallins which led us to investigate the source of orn. Here, we detected such modifications of orn in these proteins. Acid hydrolysis of arginine (arg)‐base AGE standards produced orn at different yields. The data provide unequivocal evidence for the in vivo formation of orn and its own AGEs in aging proteins, and suggest that arg‐based AGEs serve as precursors of orn.
Formation of α‐Aminoadipic and γ‐Glutamic Semialdehydes in Proteins by the Maillard Reaction
Authors: AKAGAWA MITSUGU , SASAKI DAISUKE , KUROTA YAYOI , SUYAMA KYOZO
Published: July 2005

Abstract: Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. α‐Aminoadipic semialdehyde (AAS) and γ‐glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high‐performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p‐aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA‐derivatized protein, ABA‐AAS and ABA‐GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 μM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.
Effect of Glucose Concentration on Formation of AGEs in Erythrocytes in Vitro
Authors: NAGAI RYOJI , DEEMER ELIZABETH K. , BROCK JONATHAN W. , THORPE SUZANNE R. , BAYNES JOHN W.
Published: July 2005

Abstract: Posttranslational modifications, such as advanced glycoxidation and lipoxidation end products (AGE/ALEs), are implicated in the pathogenesis of diabetic complications and atherosclerosis. Recent studies have demonstrated that AGE/ALEs are generated not only in extracellular matrix proteins, but also in intracellular proteins from metabolic intermediates. In this study we investigate the effect of glucose concentration on the formation of the AGE/ALEs, Nε‐(carboxymethyl)lysine (CML), Nε‐(carboxyethyl)lysine (CEL), S‐(carboxymethyl)cysteine (CMC), and S‐(2‐succinyl)cysteine (2SC) in erythrocytes as a function of glucose concentration. Human erythrocytes (10% hematocrit) were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 5 mM or 30 mM glucose for 5 days at 37°C. Globin was recovered by precipitation with 0.25 M HCl in acetone. Following acid hydrolysis, amino acids were converted to their trifluoroacetyl methyl ester derivatives and analyzed by GC/MS/MS. The CML and CEL content of globin increased in a time‐ and glucose‐dependent manner and also increased 1.3‐ and 1.8‐fold, respectively, in incubations containing 30 mM glucose; whereas CMC and 2SC content did not change during the five‐day incubations. Furthermore, CEL content of globin in erythrocytes incubated with 30 mM was the highest in the other AGEs, indicating that methylglyoxal may play a major role in AGE formation in erythrocytes. The erythrocyte system should be useful for cellular screening of the efficacy of inhibitors of AGE/ALE formation.
Methylglyoxal Induces Apoptosis through Oxidative Stress‐Mediated Activation of p38 Mitogen‐Activated Protein Kinase in Rat Schwann Cells
Authors: FUKUNAGA MICHIRU , MIYATA SATOSHI , HIGO SATOMI , HAMADA YASUHIRO , UEYAMA SHIGEMITSU , KASUGA MASATO
Published: July 2005

Abstract: Although recent studies have suggested the potential involvement of apoptotic cell death in the development of diabetic neuropathy, the precise mechanism remains to be elucidated. On the other hand, it is known that the formation of methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions through several glucose‐related metabolisms including the glycation reaction. We found that MG was capable of inducing apoptosis in peripheral nerve‐derived Schwann cells (SCs) in a time‐ and dose‐dependent manner, accompanied by a reduction of intracellular glutathione content. Furthermore, MG induced phosphorylation of MKK3/MKK6, an upstream molecule in the p38 MAPK pathway. N‐acetyl‐L‐cysteine, an antioxidant, successfully suppressed the activity of the p38 MAPK signaling pathway along with the inhibition of apoptosis, indicating the involvement of oxidative stress in the MG‐induced apoptosis via the p38 MAPK pathway. These results suggest a possible contribution of glucose‐derived MG to the development of diabetic neuropathy by injuring the cellular constituent of the peripheral nerve system, such as SCs, in the hyperglycemic milieu.
Dicarbonyl Stress and Apoptosis of Vascular Cells: Prevention by αB‐Crystallin
Authors: NAGARAJ RAM H. , OYA‐ITO TOMOKO , BHAT MANJUNATHA , LIU BINGFEN
Published: July 2005

Abstract: Methylglyoxal (MGO) is an alpha‐dicarbonyl compound produced from triose phosphate intermediates of glycolysis. It reacts rapidly with proteins to produce advanced glycation products. We have studied the effect of MGO modification of fibronectin on retinal capillary cell viability. Our studies show that pericytes grown on MGO‐modified fibronectin (FN) undergo enhanced apoptosis through the p38MAPK‐mediated oxidative pathway and that αB‐crystallin, a stress protein present in pericytes, can protect them from MGO‐mediated apoptosis. Our studies with vascular endothelial cells show that hyperglycemia‐induced apoptosis is inhibited by overexpression of αB‐crystallin. These observations suggest a novel role of αB‐crystallin in hyperglycemia‐mediated damage to vascular cells in diabetes.
Phototransformations of Advanced Glycation End Products in the Human Eye Lens due to Ultraviolet A Light Irradiation
Authors: ARGIROV O.K. , LIN B , ORTWERTH B J
Published: July 2005

Abstract: Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP‐lysine and argpyrimidine are two UVA‐absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP‐lysine, OP‐phenethylamine (a phenethylamine analogue of OP‐lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP‐lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP‐lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP‐lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP‐phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.
The Maillard Reaction for Sunlight Protection
Authors: FUSARO RAMON M. , RICE EDWIN G.
Published: July 2005

Abstract: During seven months of a clinical trial in spring, summer, and fall, 30 UVA/B/Soret band‐photosensitive patients used sequential topical applications of dihydroxyacetone (DHA) followed by naphthoquinone only at bedtime and received excellent photoprotection without a single therapeutic failure or loss of any patient to follow‐up. Eighteen of the 30 patients extended the limits of their photoprotection repeatedly over a seven‐month period to tolerate without sunburns six to eight hours of midday sunlight under all kinds of occupational and recreational environmental conditions. Previously, the use of 3% DHA topically in earlier studies gave only a sun protection factor (SPF) of 3. In this reanalysis of the original notes of a previous clinical study of the melanoidins produced by DHA followed by naphthoquinone in the keratin layers of the epidermis of minimally pigmented Caucasian photosensitive patients, it is determined that these patients received a minimal UVB photoprotection of SPF 18 or more. This represents at least a sixfold amplification of the UVB photoprotective effect over the use of only dihydroxyacetone in the Maillard reaction.
K2P—A Novel Cross‐Link from Human Lens Protein
Authors: CHENG RONGZHU , FENG QI , ARGIROV OGNYAN K. , ORTWERTH BERYL J.
Published: July 2005

Abstract: We report here the isolation of a novel acid‐labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC‐MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One‐ and two‐dimensional NMR analyses elucidated the structure as being 1‐(5‐amino‐5‐carboxypentyl)‐4‐(5‐amino‐5‐carboxypentyl‐amino)‐3‐hydroxy‐2, 3‐dihydropyridinium, a cross‐link between the ε‐amino groups of two lysine residues and a five‐carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water‐soluble and water‐insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 ± 362 pmol/mg of water‐insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of water‐soluble [WS] protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross‐link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.
Ketosis Leads to Increased Methylglyoxal Production on the Atkins Diet
Authors: BEISSWENGER BENJAMIN G. K. , DELUCIA ELIZABETH M. , LAPOINT NANCY , SANFORD REBECCA J. , BEISSWENGER PAUL J.
Published: July 2005

Abstract: In the popular and widely used Atkins diet, the body burns fat as its main fuel. This process produces ketosis and hence increased levels of β‐hydroxybutyrate (BOB) acetoacetate (AcAc) and its by‐products acetone and acetol. These products are potential precursors of the glycotoxin methylglyoxal. Since methylglyoxal and its byproducts are recognized as a significant cause of blood vessel and tissue damage, we measured methylglyoxal, acetone, and acetol in subjects on the Atkins diet. We found that by 14‐28 days, methylghyoxal levels rose 1.67‐fold (P= 0.039) and acetol and acetone levels increased 2.7‐ and 6.12‐fold, respectively (P= 0.012 and 0.028). Samples from subjects with ketosis showed even greater increases in methylglyoxal (2.12‐fold), as well as acetol and acetone, which increased 4.19‐ and 7.9‐fold, respectively; while no changes were seen in samples from noncompliant, nonketotic subjects. The increase in methylglyoxal implies that potential tissue and vascular damage can occur on the Atkins diet and should be considered when choosing a weight‐loss program.
Methylglyoxal, Glyoxal, and Their Detoxification in Alzheimer's Disease
Authors: KUHLA BJÖRN , LÜTH HANS‐JOACHIM , HAFERBURG DIETRICH , BOECK KATHARINA , ARENDT THOMAS , MÜNCH GERALD
Published: July 2005

Abstract: The accumulation of advanced glycation end products (AGEs) in brains with Alzheimer's disease (AD) has been implicated in the formation of insoluble deposits such as amyloid plaques and neurofibrillary tangles. AGEs are also known to activate glia, resulting in inflammation and neuronal dysfunction. As reactive intermediates of AGE formation, neurotoxic reactive dicarbonyl compounds such as glyoxal and methylglyoxal have been identified. One of the most effective detoxification systems for methylglyoxal and glyoxal is the glutathione‐dependent glyoxalase system, consisting of glyoxalase I and glyoxalase II. In this study, we have determined the methylglyoxal and glyoxal levels in the cerebrospinal fluid of AD patients compared to healthy controls. Methylglyoxal levels in AD patients were twofold higher than in controls, but this difference was not significant due to the large intergroup variations and the small sample size. However, the concentrations of both compounds were five to seven times higher in CSF than in plasma. We also investigated the glyoxalase I level in AD and healthy control brains. The number of glyoxalase I‐ positive neurons were increased in AD brains compared to controls. Our findings suggest that glyoxalase I is upregulated in AD in a compensatory manner to maintain physiological methylglyoxal and glyoxal levels.
Evaluation of Glyoxal and Methylglyoxal Levels in Uremic Patients under Peritoneal Dialysis
Authors: LAPOLLA A , FLAMINI R , LUPO A , ARICÒ N C , RUGIU C , REITANO R , TUBARO M , RAGAZZI E , SERAGLIA R , TRALDI P
Published: July 2005

Abstract: Advanced glycation end products (AGEs) accumulate in serum and tissues of patients with chronic renal failure, even in the absence of diabetes, and a different clearance of these species has been observed by hemodialysis and peritoneal dialysis (CAPD). Furthermore, it has been shown that not only AGE but also 1,2‐dicarbonyl compounds are formed during heat sterilization of glucose‐based peritoneal dialysis fluids. Therefore, we investigated the level of some AGEs (pentosidine and free pentosidine) and dicarbonyl compounds (glyoxal and methylglyoxal) in end‐stage renal disease patients subjected to peritoneal dialysis. Samples (20 from healthy subjects, 16 from uremic patients before and after 12 h of peritoneal dialysis) were analyzed, and the plasma and dialysate levels of glyoxal, methylglyoxal, pentosidine, and free pentosidine were determined. In plasma of uremic patients, mean values of pentosidine showed a small decrease after dialysis and were always higher than those of healthy control subjects. An analogous trend was observed for free pentosidine. In the case of peritoneal dialysate, no pentosidine and free pentosidine were found at time zero, whereas both compounds were detected after 12 h of dialysis. Glyoxal and methylglyoxal mean levels showed a decrease in plasma after dialysis even if their values were always higher than those of healthy control subjects. Surprisingly, an analogous trend was observed also in dialysate. These results might indicate that glyoxal and methylglyoxal already present in the dialysis fluid react with the peritoneal matrix proteins, accounting for the gradual loss of peritoneal membrane function that is often observed in patients subjected to CAPD for a long time.
Application of Semiquantitative Proteomics Techniques to the Maillard Reaction
Authors: AMES JENNIFER M.
Published: July 2005

Abstract: Proteomic tools—in particular, mass spectrometry (MS)—have advanced significantly in recent years, and the identification of proteins within complex mixtures is now a routine procedure. Quantitative methods of analysis are less well advanced and continue to develop. These include the use of stable isotope ratio approaches, isotopically labeled peptide standards, and nonlabeling methods. This paper summarizes the use of MS as a proteomics tool to identify and semiquantify proteins and their modified forms by using examples of relevance to the Maillard reaction. Finally, some challenges for the future are presented.
The Carbon Module Labeling (CAMOLA) Technique: A Useful Tool for Identifying Transient Intermediates in the Formation of Maillard‐Type Target Molecules
Authors: SCHIEBERLE PETER
Published: July 2005

Abstract: Although the Maillard reaction is a well‐known source of aroma and taste compounds in processed foods, a systematic correlation of the concentrations of most of the reaction products identified so far with human perception has scarcely been performed. Furthermore, the influence of process parameters on yields and formation mechanisms of key flavor compounds has not been systematically studied. In this short state‐of‐the‐art review, concepts to characterize flavor‐active food constituents are briefly discussed, and approaches to elucidate formation mechanisms from labeling experiments and isotopomeric quantitation are highlighted on the basis of results obtained in the author's laboratory.
Analysis of Protein Glycation Products by MALDI‐TOF/MS
Authors: KISLINGER THOMAS , HUMENY ANDREAS , PEICH CARLO C. , BECKER CORD‐MICHAEL , PISCHETSRIEDER MONIKA
Published: July 2005

Abstract: Matrix‐assisted laser desorption ionization‐mass spectrometry with time‐of‐flight detection (MALDI‐TOF/MS) is a promising tool to analyze advanced glycation end product (AGE)‐modified proteins. The combination of soft ionization (MALDI) with time‐of‐flight mass detection allows analysis of peptides and proteins of a molecular mass up to 300 kDa with minimal sample workup. Because the direct structural analysis of intact AGE proteins is not possible due to the formation of broad and poorly resolved peaks, peptide mapping was introduced into the analysis of AGE proteins by MALDI‐TOF/MS, allowing site‐specific analysis of defined AGEs. When methylglyoxal‐modified lysozyme was subjected to MALDI‐TOF/MS peptide mapping, methylimidazolone and argpyrimidine attached to the arginine residue and carboxyethyl (CEL) bound to the lysine were detected on peptideaa1‐7 (KVFGRCE). In contrast, only one methylimidazolone was found on peptideaa8‐35 (LAAAMKRHGLDNYRGYSLGNWVCAAKFE) and peptideaa120‐129 (VQAWIRGCRL), respectively. The analysis of AGE protein, which had been incubated with glucose, revealed the presence of an Amadori product and a carboxymethyl residue (CML) on peptideaa1‐7 and peptideaa8‐35, as well as an imidazolone A on peptideaa120‐129. Furthermore, the early Maillard reaction of lysozyme, which had been glycated by seven different sugars, was monitored by MALDI‐TOF/MS peptide mapping. Finally, this approach was successfully applied for site‐ and product‐specific relative quantification of AGEs. For example, kinetics of CML and Amadori product formation on peptideaa1‐7, as well as imidazolone A formation on peptideaa120‐129, were determined.