Degradation of L-Ascorbic Acid and Mechanism of Non-enzymic Browning Reaction

In the presence of oxygen, L-ascorbic acid sol ution (0.05 M) browned more intense1 y than dehydro-L-ascorbic acid solution (0.05 M) during storage for longer period.

The mixed solution of L-ascorbic acid (ASA) and dehydro-L-ascorbic acid (DHA) with the ratio of 1:1 or 1:3 in concentration gave more intense browning than DHA solution during storage at 38°C for about 3 weeks. Essentially the same type of browning was observed in case of the mixture of ASA and DHA with D-glucose. Browning of partially oxidized ASA solution also showed substantially the same results as those mentioned above.     

Formation of 1-Deoxy-d-threo-2,5-Hexodiulose by Radiation Induced Chain Reaction in Crystalline d-Fructose

Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2×10^-9 M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIIa) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.     

Studies on Antioxidant Activity of Nonenzymic Browning Reaction Products

A mixture of 0.8 M D-xylose and 0.8 M glycine, when heated at 100°C, showed inhibitory effect against autoxidation of 40% ethanol solution of linoleic acid. The antioxidant activity increased in proportion to color intensity of browning reaction solution, whereas reductones formed during the browning process showed little contribution to the activity. Nondialyzable melanoidin fraction of browning solution also showed a positive activity. Consequently, it was considered that melanoidin pigment would play an important role in the antioxidant activity.     

真核细胞中mRNA的降解机制

细胞中不同的ｍ ＲＮＡ 半寿期差异很大,ｍ ＲＮＡ 的稳定性受到多种因素的影响,现在已经发现了许多对ｍ ＲＮＡ 的稳定性有影响的顺式因子和反式因子。大量的研究证明在真核细胞内存在复杂的机制调节ｍ ＲＮＡ 的稳定与降解及其所引起的基因表达。现在可以肯定在真核细胞中至少存在着三种ｍ ＲＮＡ的降解方式：依赖于脱腺苷酸的降解,无义密码介导的ｍ ＲＮＡ 的降解和核酸内切酶的水解。其中依赖于脱腺苷酸的降解方式是细胞内大多数ｍ ＲＮＡ 降解的主要途径。     

Mechanisms of Protein Degradation: An Odyssey with ODC

Intracellular proteolysis plays an important role in regulating fundamental cellularprocesses such as cell cycle, immune and inflammation responses, development,differentiation, and transformation. The ubiquitin-proteasome system accounts for thedegradation of the majority of cellular short-lived proteins. This system involves theconjugation of multiple ubiquitin residues to the target protein and its recognition by the26S proteasome through the poly-ubiquitin chain. Studies on the degradation of ornithinedecarboxylase (ODC) demonstrated that poly-ubiquitin is not the only signal recognizedby the 26S proteasome. The recognition of ODC by the 26S proteasome is mediated byinteraction with a polyamine-induced protein termed, antizyme (Az). While thedegradation of ODC is ubiquitin-independent, the degradation of its regulator Az, and ofantizyme-inhibitor (AzI), an ODC homologous protein that regulates Az availability, areubiquitin dependent. Interestingly, ODC undergoes another type of ubiquitin-independentdegradation by the 20S proteasome that is regulated by NAD(P)H quinoneoxidoreductase 1 (NQO1). Considering the prevalence of the ubiquitin system in theprocess of cellular protein degradation it is rather remarkable that a key cellular enzymeis subjected to two different proteolytic pathways that are different from the ubiquitindependent one. This exceptional behavior of ODC provides us with valuable insightsregarding protein degradation in general.     

Possibility of the Nonenzymatic Browning (Maillard) Reaction in the ISM

The possibility of the occurrence of the nonenzymatic browning reaction in the gaseous phase in the interstellar medium has been investigated by using Density Functional Theory computations. Mechanisms for the reactions between formaldehyde (Fald) + glycine (Gly), Fald + NH ₃ and Fald + methylamine (MeAm) have been proposed, and the possibility of the formation of different compounds in the proposed mechanisms has been evaluated through calculating the Gibb's free energy changes for different steps of the reaction, by following the total mass balance. The Fald + Gly reaction under basic conditions is found as the most favorable for producing 1-methyl-amino methene or 1-methyl-amino methelene (MAM). The reaction under acidic conditions is found to be the least favorable for producing MAM. The Fald + NH ₃ reaction is found to be plausible for the production of MeAm, which can participate by reaction with Fald, resulting in the formation of MAM.     

Inhibition of Bacillus megaterium by a Trimethylamine Oxide-Associated Browning Reaction Product

Heated combinations of trimethylamine oxide (TMAO) and culture media (tryptone, glucose, yeast extract broth or a defined minimal medium), or heated TMAO and glucose, contained substance(s) that inhibited growth of Bacillus megaterium. Inhibition was expressed primarily as an increase of the lag phase of growth; the logarithmic growth rate was comparable to control cultures. The addition of unheated TMAO to the culture media had no effect on growth. Results suggested that TMAO was decomposed during heating and that dimethylamine, one of the degradation products, reacted with glucose by a Maillard-Amadori reaction to produce the inhibitory substance(s).     

Formation of Two-Carbon Sugar Fragment at an Early Stage of the Browning Reaction of Sugar with Amine

A product easily converted to glyoxal was found in an early stage of the reaction of sugar with amine in ethanol. Glyoxaldicyclohexylimine was isolated from the reaction mixture of d-glucose with cyclohexylamine. This finding suggested the formation of a similar type glyoxaldialkylimine in other reactions of sugar with amine. This two-carbon compound was assumed to be produced directly from sugar or glycosylamine, and a new pathway for sugar fragmentation was proposed.     

Oxidation of Free Methionine and Methionine Residues in Protein Involved in the Browning Reaction of Phenolic Compounds

The oxidation of methionine to its sulfoxide, as a possible cause of decrease in the biological value of red clover during drying with aeration, was examined using various model systems, in the presence or absence of polyphenol oxidase. The effects of catalase were also examined. Results indicated hydrogen peroxide as a possible intermediate that directly oxidizes methionine. The methionine oxidation can be one of the causes of the decrease in biological value of red clover during drying, beside the known damage of lysine in the same process.     

Mechanisms of Hop Inhibition Include the Transmembrane Redox Reaction

In this work, a novel mechanistic model of hop inhibition beyond the proton ionophore action toward (beer spoiling) bacteria was developed. Investigations were performed with model systems using cyclic voltammetry for the determination of redox processes/conditions in connection with growth challenges with hop-sensitive and -resistant Lactobacillus brevis strains in the presence of oxidants. Cyclic voltammetry identified a transmembrane redox reaction of hop compounds at low pH (common in beer) and in the presence of manganese (present in millimolar levels in lactic acid bacteria). The antibacterial action of hop compounds could be extended from the described proton ionophore activity, lowering the intracellular pH, to pronounced redox reactivity, causing cellular oxidative damage. Accordingly, a correlation between the resistance of L. brevis strains to a sole oxidant to their resistance to hop could not be expected and was not detected. However, in connection with our recent study concerning hop ionophore properties and the resistance of hop-sensitive and -tolerant L. brevis strains toward proton ionophores (J. Behr and R. F. Vogel, J. Agric. Food Chem. 57:6074-6081, 2009), we suggest that both ionophore and oxidant resistance are required for survival under hop stress conditions and confirmed this correlation according to the novel mechanistic model. In consequence, the expression of several published hop resistance mechanisms involved in manganese binding/transport and intracellular redox balance, as well as that of proteins involved in oxidative stress under “highly reducing” conditions (cf. anaerobic cultivation and “antioxidative” hop compounds in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress-induced mechanisms. Beyond this specific model of hop inhibition, these investigations provide general insight on the role of electrophysiology and ion homeostasis in bacterial stress responses to membrane-active drugs.The inflorescences of the hop plant Humulus lupulus are traditionally used for beer brewing, due to the pleasant bitterness of hop compounds in beer and their additional inhibitory effect on bacteria. These antibiotic and bacteriostatic properties of hops comprise several inhibitory mechanisms. The described effects of hop compounds on bacteria are permeability changes in the bacterial cell wall (30), leakage of the cytoplasmic membrane and a subsequent inhibition of respiration, and protein, DNA, and RNA synthesis (42), as well as changes in leucine uptake and proton ionophore activity (33). In a recent study (5), we characterized the latter ionophore properties of hop compounds in a cell-free model system via bilayer lipid membrane (BLM) techniques in connection with growth challenges of hop-sensitive and -resistant Lactobacillus brevis strains. The antibacterial action of iso-α-acids as proton ionophores could be confirmed by the BLM measurements; however, that the reported ionophore properties enable electroneutral H⁺/Mn²⁺ exchange (34) could not be verified. On the other hand, the measurements indicated the manganese-dependent enhancement of transmembrane charge permeation. The origin of high membrane potential formation in the presence of manganese, as well as strongly elevated membrane conductivity with a concomitant increase in the effectiveness of an uncoupler, suggests a different origin of charge transfer under these conditions. Accordingly, the expected cross-resistance to sole proton ionophores could not be detected (5), and any antibiotic tested until now could not simulate the stress induced by hop compounds in bacteria (11). The latter observation may be related to the complexity of the chemical composition of hops, which is due to compound variations resulting from variations in the natural product as well as to consecutive chemical conversions of hop compounds, which are known to be highly reactive. This high level of reactivity of hop compounds comprises polymerization as well as reduction and oxidation processes (6, 36). The latter are also known to efficiently inhibit bacteria by causing oxidative stress, in which oxidizing molecules are generated at a higher rate than they are detoxified. To counteract oxidative stress (7, 12, 20, 21, 41, 44, 46), bacteria developed several defense strategies, namely, reduced generation of oxidizing molecules during metabolism, enzymatic or nonenzymatic detoxification of the latter, and repair of damaged cell components (20). In this context, the importance of the nonenzymatic detoxification mechanism of oxidative molecules in lactic acid bacteria is connected to their extraordinary high levels of intracellular manganese (1). Mn²⁺ can cycle in vivo between the oxidation states +2 and +3, whereas the ligand environment alters the redox potential. Thus, “activated” Mn²⁺ can act as a scavenger of toxic oxygen species (17), whereas Mn³⁺ and its complexes are able to oxidize different substrates and contribute to the overall oxidative potential (24). However, in the presence of hop compounds, hop-resistant L. brevis TMW 1.465 strongly reduced intracellular manganese levels (normally elevated under environmental stress conditions [17]) and changed the protein expression profile, leading to an elevated rate of manganese binding and/or manganese-dependent enzymes as well as to the expression of repair proteins and proteins of membrane lipid synthesis, which counteract oxidative damage (3, 4). This unusual regulation could possibly be linked to the fact that the ligand environment (e.g., iso-α-acids [34]) alters the redox properties of manganese. On the other hand, the possibility cannot be excluded that the redox properties of hop compounds, which are known to be highly redox-reactive substances (6), are altered in the presence of manganese. Accordingly, a study of the redox reactivity of hop compounds in connection with divalent manganese is required.An appropriate method to investigate possible oxidation processes induced by hop compounds and their manganese complexes (34, 37) is cyclic voltammetry (voltamperometry). This method can be applied in aqueous solutions (28, 39) as well as upon reconstituted planar bilayer lipid membranes (14-16, 29). The use of cyclic voltammetry for determination of redox processes/conditions as described for hop compounds containing beer samples has considerable advantages compared to potentiometric measurements, as it is fast and reproducible (39). Further, cyclic voltammetry is an elegant tool for the study of transmembrane redox reactions in electron-conducting BLMs. These transmembrane redox processes play a central role in life''s membrane bioenergetics (43). The possibility of the application of cyclic voltammetry to a BLM resides in the double-electrode behavior of the planar membrane (8). The electron transport process through the BLM implies electron transfers at both solution-membrane interfaces, as well as an electron transfer through the BLM. Consequently, redox couples taking part in the process must be located at the membrane-solution interfaces as well as inside the membrane in their oxidized and reduced form. These redox couples are not necessarily the same in solutions and membranes. Nevertheless, for transmembrane electron transfer, proximity of the redox potentials of both is required (29). As lactic acid bacteria can contain millimolar concentrations of membrane-impermeable divalent manganese located near the cytoplasmic membrane (1) and hop compounds are amphiphilic compounds, a great deal of the above-described assumptions required for transmembrane redox processes is fulfilled. Consequently, redox characteristics of membrane-active hop-derived substances were investigated, the results of which can undoubtedly contribute to the understanding of biological membrane processes under hop stress conditions (14).     

Mutagenicity of Intermediates Produced in the Early Stage of the Browning Reaction of Triose Reductone with Nucleic Acid Related Compounds on Bacterial Tests

Two types of reductive intermediates, linear and tricyclic forms, isolated from browning mixtures of triose reductone (TR) with guanine and its derivatives showed evident mutagenicity on Salmonella typhimurium TA 100 without S-9 mixture. The linear intermediates, N²-(3-oxo-2-hydroxypropenyl) compounds of guanine, guanosine, 2′(3′)-guanylic acid and 5′-guanylic acid were more effective than the tricyclic one, l, N²-(2-hydroxypropenylidene)guanine, though they were far less active than 4-nitroquinoline-N-oxide. No acceleration in mutagenicity was observed with Cu^{2 +} and other metal ions. The reaction mixtures of TR and nucleic acid bases were also mutagenic on TA 100. Intermediates of TR with guanine and its derivatives did not have a lethal effect in Recassays with Bacillus subtilis.