Development of a Highly Selective Two-Photon Probe for Methylglyoxal and its Applications in Living Cells, Tissues, and Zebrafish.

Methylglyoxal (MGO) is one of the most important active carbonyl compounds in living organisms. It is a metabolic by product of glycolysis. MGO participates in glycosylation of proteins and nucleic acids to trigger carbonyl stress, inducing pathological status and even exacerbateing the development of chronic degenerative diseases. In order to study the diseases caused by MGO, it is meaningful for us to develop methods that could efficiently detect MGO. In our work, a new two-photon fluorescent turn-on probe which named NP has been designed which was made up of naphthalimides dye as the two-photon fluorescent platform and the o-phenylenediamine as recognition site. When reacted with MGO, NP showed excellent sensitivity and selectivity. Based on the two-photon fluorescence imaging technology, NP has firstly successful application in living cells, tissues and zebrafish to detecting MGO.

Product Studies and Mechanistic Analysis of the Reaction of Methylglyoxal with Deoxyguanosine.

Methylglyoxal (MG) is a highly reactive electrophile produced endogenously as a byproduct of glucose metabolism and protein catabolism and exogenously as a food contaminant. MG reacts spontaneously with proteins, lipids, and nucleic acids to form advanced glycation end products (AGEs), modifying or inhibiting their function. Protein AGEs are associated with pathological complications of diabetes, cancer, and neurodegenerative diseases, while the physiological impact of DNA, RNA, and lipid AGE formation is less well explored. Conflicting reports in the literature on the biologically significant DNA-AGE product distribution and mechanisms of formation prompted a re-examination of the reaction products of MG with dG, oligonucleotides, and plasmid DNA under varying conditions of MG:dG stoichiometry, pH, and reaction time. Major products identified using sequential mass fragmentation and authentic standards were N2-(1-carboxyethyl)-2'-dG (CEdG), N2-(1-carboxyethyl)-7-1-hydroxy-2-oxopropyl-dG (MG-CEdG), and 1,N2-(1,2-dihydroxy-2-methyl)ethano-2'-dG (cMG-dG). CEdG and MG-CEdG were observed in all DNA substrates, although cMG-dG was not detected to any significant extent in oligomeric or polymeric DNA. Product analyses of reactions under conditions of diminished water activity as well as results from H218O labeling indicated that MG hydration equilibria plays an important role in controlling product distribution. In contrast to previous reports, our data support independent mechanisms of formation of CEdG and cMG-dG, with the latter kinetic product undergoing reversible formation under physiological conditions.

Pathophysiological insights of methylglyoxal induced type-2 diabetes.

Diabetes mellitus is a metabolic disorder constituting a major health problem whose prevalence has gradually increased worldwide over the past few decades. Type 2 diabetes mellitus (T2DM) remains more complex and heterogeneous and arises as a combination of insulin resistance and inadequate functional ß-cell mass and comprises about 90% of all diabetic cases. Appropriate experimental animal models are essential for understanding the molecular basis, pathogenesis of complications, and the utility of therapeutic agents to abrogate this multifaceted disorder. Currently, animal models for T2DM are obtained as spontaneously developed diabetes or diabetes induced by chemicals or dietary manipulations or through surgical or genetic methods. The currently used diabetogenic agents have certain limitations. Recently, methylglyoxal (MG), a highly reactive compound derived mainly from glucose and fructose metabolism has been implicated in diabetic complications. MG is a major precursor of the advanced glycation end product (AGE) and promotes impaired functions of insulin signaling, GLUT transporters, anion channels, kinases, and endothelial cells and is finally involved in apoptosis. Recent array of literature also cited that higher concentrations of MG causes rapid depolarization, elevated intracellular Ca(2+) concentration, and acidification in pancreatic ß-cells. This review henceforth highlights the mechanism of action of MG and its implications in the pathophysiology of experimental diabetes.

Formation of 4(5)-methylimidazole and its precursors, α-dicarbonyl compounds, in Maillard model systems.

Glyoxal, methylglyoxal, and diacetyl formed from sucrose alone and from a D-glucose/ammonia Maillard model system were analyzed by gas chromatography. They are known as precursors of 4(5)-methylimidazole (MI). Glyoxal and methylglyoxal formed more in acidic conditions than in basic conditions, whereas diacetyl formed the most at the highest basic condition of pH 12. Glyoxal formation from sucrose ranged from 0.33 to 32.90 µg/g under four different time and temperature conditions. Amounts of glyoxal, methylglyoxal, and diacetyl formed in Maillard model systems ranged from 2.98 to 46.12 µg/mL, from 8.27 to 156.61 µg/mL, and from 14.94 to 1588.45 µg/mL, respectively. 4(5)-MI formation in the same model systems ranged from 28.56 to 1269.71 µg/mL. Addition of sodium sulfite reduced formation of these chemicals significantly. Total α-dicarbonyl compounds in 12 commercial soft drinks ranged from 5.75 to 50.72 µg/mL. 4(5)-MI was found in levels ranging from 1.76 to 28.11 ng/mL in 10 commercial soft drinks.

Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey.

Dihydroxyacetone (DHA) and methylglyoxal (MGO) are unique carbohydrate metabolites of manuka honey. A method for the reliable quantification of DHA in honey samples was established, based on derivatization with o-phenylenediamine (OPD) and subsequent RP-HPLC with UV detection. The previously unknown reaction product of DHA and OPD was identified as 2-hydroxymethylquinoxaline by spectroscopic means. DHA was exclusively determined in 6 fresh manuka honeys originating directly from the beehive as well as 18 commercial manuka honey samples, ranging from 600 to 2700 mg/kg and 130 to 1600 mg/kg, respectively. The corresponding MGO contents varied from 50 to 250 mg/kg in fresh and 70 to 700 mg/kg in commercial manuka honey samples. A good linear correlation between DHA and MGO values in commercial manuka honeys was observed, resulting in a mean ratio of DHA to MGO of 2:1. In contrast to this, the DHA-to-MGO relation was much higher in fresh manuka honeys but approximated to a ratio of 2:1 while honey ripening. Heating experiments revealed that MGO formation based on thermal treatment as a consequence, for example, of caramelization in honey does not occur. DHA and MGO can serve as suitable unique quality parameter for manuka honey.

Design, synthesis and biological evaluation of 3-(4-halophenyl)-3-oxopropanal and their derivatives as novel antibacterial agents.

In continuing our program aimed to search for potent drugs for bacterial infections, a series of 3-(4-halophenyl)-3-oxopropanal and their derivatives were designed, synthesized and their antibacterial activities in vitro against both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa were evaluated. Compounds 7, 8, 13-16, 21 and 22 had moderate antibacterial activities against Staphylococcus aureus (minimal inhibitory concentration (MIC) <16 microg/ml), suggesting that the introduction of mono-methoxyamine or ethoxyamine moiety might play an important role in determining the potent antibacterial activities. Furthermore, the antibacterial activities of select compounds 7, 15 and 16 against the clinically important pathogenic bacteria-methicillin-resistant Staphylococcus aureus (MRSA) were also investigated. Results showed that these compounds exhibited more potent activities than the well-known antibacterial agents Houttuynin and Levofloxacin.

Regio- and chemoselective multiple functionalization of chloropyrazine derivatives. Application to the synthesis of coelenterazine.

Successive regio- and chemoselective metalations of chloropyrazines using TMPMgCl x LiCl and TMPZnCl x LiCl furnish, after trapping with electrophiles, highly functionalized pyrazines in high yields. Application to a synthesis of coelenterazine, a bioluminescent natural product in jellyfish Aequorea victoria, in nine steps (9% overall yield) is reported.

Spectroscopic studies of methylglyoxal in water and dimethylsulfoxide.

Methylglyoxal is a highly reactive dicarbonyl compound, which reacts in vivo with biological macromolecules and thereby affects their structure and function. These changes are associated with complications during aging, diabetes and Alzheimer's disease as well as with growth inhibition in different tumors. Many enzymes are involved in the metabolism of methylglyoxal, but its true physiological role in metabolism and chemical properties are still obscure. In this study it was shown that methylglyoxal, during the freeze-drying of aqueous solutions, polymerizes into small polymeric structures which are stable in organic media such as dimethylsulfoxide. When re-exposed to water, the polymers are immediately transformed into the monomeric mono- and dihydrate forms of methylglyoxal. By NMR and UV spectroscopy, it was shown that solvent, temperature, and the amount of available water strongly influence the equilibrium of the different forms of methylglyoxal and thereby change its reactivity. 1H and 13C NMR spectroscopy were used to determine the structures of the different monomeric and oligomeric structures of methylglyoxal.

The sugar model: catalytic flow reactor dynamics of pyruvaldehyde synthesis from triose catalyzed by poly-l-lysine contained in a dialyzer.

The formation of pyruvaldehyde from triose sugars was catalyzed by poly-l-lysine contained in a small dialyzer with a 100 molecular weight cut off (100 MWCO) suspended in a much larger triose substrate reservoir at pH 5.5 and 40 degrees C. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35 mL) within the dialyzer generated pyruvaldehyde with a synthetic intensity (rate/volume) that was 3400 times greater than that of the triose substrate solution (12 mM, 120 mL) outside the dialyzer. Under the given conditions the final yield of pyruvaldehyde was greater than twice the weight of the polylysine catalyst. During the reaction the polylysine catalyst was poisoned presumably by reaction of its amino groups with aldehyde reactants and products. Similar results were obtained using a dialyzer with a 500 MWCO. The dialyzer method of catalyst containment was selected because it provides a simple and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.

A new class of synthetic biological response modifiers: the methylfurylbutyrolactones (Nafocare B).

A new class of synthetic biological response modifiers (BRMs) has been produced by combining a highly electrophilic reactant, 2-methyl-2, 5-dihydrofuran (a cyclic acetal of cis-3-acetyl acrolein), with L-ascorbic acid. The parent class of compounds can be referred to as methylfurylbutyrolactones (MFBL), previously termed Nafocare B. This parent molecule is amorphous, has a molecular weight of 252.7, and the chemical name [3,6] cyclohemiketal of 2-(5-methyl-2-furyl)-3-keto-L-butyrolactone. Two crystalline forms were produced by a reaction of the MFBL parent molecule with either succinic anhydride or succinimide, to create MFBL-SA (Nafocare B2) and MFBL-S (Nafocare B3) dimers, respectively. The structure of these compounds has been confirmed by modern methods of analytical chemistry, including x-ray crystallography. All three forms of the MFBLs showed negligible toxicity in single-dose acute LD-50s in mice. Also, the MFBLs did not demonstrate genotoxic activity at 800 mg/kg in the mouse micronucleus assay. The MFBLs are immunostimulatory in assays involving T- and B-lymphocytes, but not in immunoassays on macrophages derived from resident- or thioglycollate-elicited peritoneal exudate cells (PEC). Spleen cells from mice treated 4 days via the intraperitoneal, intravenous, or the oral routes responded significantly over controls to suboptimal stimulatory concentrations of polyclonal mitogens in the lymphocyte stimulation assay. The MFBLs were not mitogenic, since they did not increase DNA synthesis in resting spleen cells from MFBL-treated mice. Antibody production is also amplified by the MFBLs. Mice immunized with sheep erythrocytes, a T-cell-dependent antigen, and treated with MFBLs had a 200-800% increase in the numbers of antibody-producing splenic lymphocytes detected by the Jerne hemolytic plaque assay. Also, mice immunized with soluble bovine serum albumin (BSA), and treated with a MFBL, demonstrated at least a fourfold increase in IgG-specific antibodies to BSA, when compared with controls. To demonstrate effects of MFBLs on macrophages, we used the Fc receptor (FcR) surface marker and superoxide anion assays. Only at the highest in vitro dose of MFBL (16 micrograms/ml) was a significant increase in erythrocyte antibody rosette formation detected, using resident macrophages isolated from PEC. In the superoxide anion release assay neither resident- nor thioglycollate-elicited PECs, obtained from in vivo-treated mice or macrophages treated in vitro, showed increased production of superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)