Aggregation as a consequence of glycation: insight into the pathogenesis of arthritis.

Advanced glycation end products (AGEs) as a result of Maillard reaction are currently at the heart of the pathogenesis of several diseases and hence are the objective of numerous investigations. Glycation of proteins has been an implication in long-term complications. Collagen is the most abundant protein in the human body. The purpose of this study was to monitor and characterize the oligomeric aggregates and AGEs of human collagen on addition of glyoxal using ultraviolet, fluorescence, circular dichroism (CD) spectroscopy, docking studies, ITC, and microscopy. Collagen was incubated for varying time periods up to 21 days with three different concentrations (5, 20, and 40 mM) of glyoxal. Collagen exists as molten globule at day 6, evident from native-like secondary structure, altered tryptophan, and high ANS fluorescence due to surface-exposed hydrophobic residues. Glycated collagen as AGEs and aggregates was observed at day 18 and 21, respectively. Formation of AGE and aggregates were confirmed by UV and fluorescence spectroscopy. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine) forming the Schiff base, the carboxymethyl lysine, and carbonyl content. Non-tryptophan fluorescence for AGEs was also monitored as the emission peak at 400 and 440 nm, respectively. SEM and TEM confirmed the oligomeric nature of aggregates. Glyoxal at 40 mM shows maximum alterations in protein structure followed by 20 and 5 mM concentration. In the present paper, we propose that a high concentration of glyoxal for a prolonged time results in the formation of harmful aggregates and AGEs.

How to help the skin cope with glycoxidation.

BACKGROUND: Protein glycation refers to the spontaneous reaction of reducing sugars with proteins and the subsequent formation of stable advanced glycation end products (AGEs). Glycation is linked with oxidative stress, and this association is called "glycoxidation". Glycoxidation alters the protein structure and function and causes tissue aging, as seen in human skin. Therefore, research on substances inhibiting glycoxidation appears to be crucial in the prevention of skin aging. With this aim, several plant extracts have been screened for antiglycation activity, and the results of the best candidates are presented in this article. METHODS: Glycation was studied on human skin proteins (collagen, elastin, and albumin) and on a model of reconstructed skin. Oxidative stress has been addressed by testing the copper-induced low-density lipoprotein oxidation, ultraviolet irradiation of glycated dermis, and carbonyl activation of human dermal fibroblasts. A clinical test evaluated the extent of oxidative stress induced by ultraviolet A irradiation. RESULTS: Among the tested products, several plant extracts have decreased the glycation effects on skin proteins collagen, elastin, and albumin. In addition, a plant extract has significantly inhibited the different forms of oxidative stress associated with protein glycation. CONCLUSIONS: We have demonstrated that plant extracts can relieve the deleterious effects of glycation on human skin. Moreover, a plant extract rich in antioxidant molecules has also significantly preserved the human skin from glycoxidation attacks.

Effect of coffee filtrate, methylglyoxal, glyoxal, and caffeine on Salmonella typhimurium and S. enteritidis survival in ground chicken breasts.

The antimicrobial effect of roasted coffee filtrate (CF) and dicarbonyls on Salmonella Typhimurium and Salmonella Enteritidis in raw ground chicken breast meat (GCB) was investigated. Coffee was brewed and filtered before addition to GCB. Coffee filtrate with and without added caffeine, methylglyoxal, and/or glyoxal was added to GCB and then inoculated with Salmonella Typhimurium and Salmonella Enteritidis. Ground chicken samples were stomached with peptone water at days 1, 3, 5, and 7, plated on XLD agar with a TSA overlay, and Salmonella survivors were enumerated. CF alone gave less than a 1 Log reduction in all runs compared to control GCB with no treatment. Methylglyoxal (2.28 mg/g GCB) had the greatest antimicrobial effect against Salmonella Typhimurium and Salmonella Enteritidis in GCB with average Log reductions of 2.27 to 3.23, respectively, over the 7 d duration of the experiment compared to control GCB with no treatment. A 1 Log reduction was observed in GCB with CF, 0.93 mg glyoxal, and 1 mg caffeine/g chicken compared to the control and GCB with only CF. Heat-produced coffee compounds could potentially reduce Salmonella in retail ground chicken and chicken products.

Isolation, identification, and quantification of roasted coffee antibacterial compounds.

Coffee brew is a widely consumed beverage with multiple biological activities due both to naturally occurring components and to the hundreds of chemicals that are formed during the roasting process. Roasted coffee extract possesses antibacterial activity against a wide range of microorganisms, including Staphylococcus aureus and Streptococcus mutans, whereas green coffee extract exhibits no such activity. The naturally occurring coffee compounds, such as chlorogenic acids and caffeine, cannot therefore be responsible for the significant antibacterial activity exerted by coffee beverages against both bacteria. The very low minimum inhibitory concentration (MIC) found for standard glyoxal, methylglyoxal, and diacetyl compounds formed during the roasting process points to these alpha-dicarbonyl compounds as the main agents responsible for the antibacterial activity of brewed coffee against Sa. aureus and St. mutans. However, their low concentrations determined in the beverage account for only 50% of its antibacterial activity. The addition of caffeine, which has weak intrinsic antibacterial activity, to a mixture of alpha-dicarbonyl compounds at the concentrations found in coffee demonstrated that caffeine synergistically enhances the antibacterial activity of alpha-dicarbonyl compounds and that glyoxal, methylglyoxal, and diacetyl in the presence of caffeine account for the whole antibacterial activity of roasted coffee.

Kinetic analysis on the sensitivity of glucose- or glyoxal-induced LDL glycation to the inhibitory effect of Psidium guajava extract in a physiomimic system.

Experimentation with a physiomimic system and kinetic analysis exhibited four distinct reaction phases in LDL glycation despite of the type of inducer: glucose or glyoxal. LDL glycation was more sensitive to a status of hyperglycemia (such as 400 mg glucose/100 mL) as evidenced by the reaction order of 0.53. Glucose reacted intensively in the Initial Phase (reaction period 0-2h) which was identified to result from a parallel mechanism involving both the direct Schiff's product formation and the auto-oxidative cleavages. In contrast, a physiological level of glyoxal revealed merely a reaction order of only 0.09, implicitly indicating a far less sensitive glycation which can be attributed to a mechanism proceeding simply through a molecular Schiff's reaction. On treatment with Psidium guajava L. aqueous extract (PE) (0.01-0.625 mg/mL), a rather unique and significant inhibitory characteristic on LDL glycation was observed with a dose-dependent manner. We attributed such an effect of PE to its distinct abundance of polyphenolic content (165.61+/-10.39 mggallic acid equivalent (GAE)/g). Conclusively, PE is an excellent anti-LDL glycative agent whose potential therapeutic uses can be extended to the prevention of a variety of cardiovascular and neurodegenerative diseases associated with glycations.

Inhibitory effect of some selected nutraceutic herbs on LDL glycation induced by glucose and glyoxal.

Anti-LDL glycative agents were investigated using aqueous extracts of Psidium guajava L. (PE), Toona sinensis Roem. (TE), Momordica charantia L. (ME) and Graptopetalum paragugayene E. Walther (GE). Concentrations of extracts 0.01-0.625 mg/mL, low density lipoprotein (LDL; 100 microg protein/mL) and inducers glucose (400 mM) and glyoxal (2.5 mM) were incubated at 37 degrees C. Evaluation parameters involved the thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), relative electrophoretic mobility (REM), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capability and total polyphenolic content. Results for anti-TBARS efficiency (in%) were PE (75.77), TE (75.10), ME (68.81) and GE (19.81) at 0.5 mg/mL, respectively, when induced by glucose; 36.68, 35.60, 32.62 and inactive, respectively, by glyoxal. The lag times for CD formation (in min) were: 289 and 125 by PE and TE, respectively, comparing to the control (45). REM was 1.6 with respect to PE (0.1 mg/mL) compared to the control (4.2). PE at 0.01 mg/mL effectively inhibited with 63.45% efficiency on AGEs induced by glucose. We conclude that PE virtually is a potent antiglycative agent, which can be of great value in the preventive glycation-associated cardiovascular and neurodegenerative diseases.

Synthesis of potato virus X RNAs by membrane-containing extracts.

Membrane-containing extracts isolated from tobacco plants infected with the plus-strand RNA virus, potato virus X (PVX), supported synthesis of four major, high-molecular-weight PVX RNA products (R1 to R4). Nuclease digestion and hybridization studies indicated that R1 and R2 are a mixture of partially single-stranded replicative intermediates and double-stranded replicative forms. R3 and R4 are double-stranded products containing sequences typical of the two major PVX subgenomic RNAs. The newly synthesized RNAs were demonstrated to have predominantly plus-strand polarity. Synthesis of these products was remarkably stable in the presence of ionic detergents.

Study of the causes of direct-acting mutagenicity in coffee and tea using the Ara test in Salmonella typhimurium.

The mutagenic activities of 6 of the chemicals identified in coffee solutions were assayed with the Salmonella Ara test, under experimental conditions optimized for coffee mutagenicity. Caffeine was the only non-mutagenic compound. Among the other 5 chemicals, hydrogen peroxide was the strongest mutagen and chlorogenic acid the weakest; methylglyoxal, glyoxal and caffeic acid exhibited intermediate mutagenicities. The minimal mutagenic doses of these components correlated negatively with their relative concentrations in coffee. It was concluded that chlorogenic acid, caffeic acid, glyoxal and methylglyoxal cannot contribute alone to the mutagenicity of coffee in the Ara test, since their minimal mutagenic concentrations were much higher than their respective levels in the coffee samples assayed. By contrast, 40-60% of the mutagenic activity in coffee and also in tea could be attributed to their H2O2 contents. Catalase abolished more than 95% of the mutagenic activity of coffee, as detected by the Ara test. A similar sensitivity to catalase has been reported by other authors in relation to the coffee mutagenicity identified by the Salmonella His test. Nevertheless, the results presented in this paper suggest that the Ara forward and the His reverse mutation tests are sensitive to the mutagenicity of different constituents in coffee solutions. We propose that the His test, sensitive at high coffee doses, mainly recognizes the mutagenicity of methylglyoxal, whilst the Ara test, sensitive at low coffee doses, mainly detects the mutagenic activity of hydrogen peroxide. The data reported also suggest that the direct-acting mutagenicity(ies) detected by the Ara test in tea solutions is (are) based on similar, if not identical, mechanisms.