Brain region-specific gene expression activation required for reconsolidation and extinction of contextual fear memory.

During fear conditioning, animals learn an association between a previously neutral or conditioned stimulus (CS) and an aversive or unconditioned stimulus (US). Subsequent reexposure to the CS alone triggers two competing processes. Brief reexposure to the CS initiates reconsolidation processes that serve to stabilize or maintain the original CS-US memory. In contrast, more prolonged reexposure to the CS leads to the formation of an inhibitory extinction (CS-no US) memory. Previous studies have established that both reconsolidation and extinction require gene expression. Consistent with this, here we first show that genetic disruption of cAMP-responsive element-binding protein (CREB)-mediated transcription blocks both reconsolidation and long-term extinction of contextual fear memory. We next asked whether reconsolidation and extinction engage CREB-mediated transcription in distinct brain regions. Accordingly, we used immunohistochemical approaches to characterize the activation of the transcription factor CREB [as well as the expression of the CREB-dependent gene Arc (activity-regulated cytoskeleton-associated protein)] after brief versus prolonged reexposure to a previously conditioned context. After brief reexposure, we observed significant activation of CREB-mediated gene expression in the hippocampus and amygdala. In contrast, after the prolonged reexposure, we observed significant activation of CREB-mediated gene expression in the amygdala and prefrontal cortex. Finally, we showed that blocking protein synthesis in either the hippocampus or the amygdala blocked reconsolidation of contextual fear memory, whereas similar blockade in the amygdala and prefrontal cortex prevented the formation of extinction memory. These experiments establish that reactivated contextual fear memories undergo CREB-dependent reconsolidation or extinction in distinct brain regions.

Upregulation of calcium/calmodulin-dependent protein kinase IV improves memory formation and rescues memory loss with aging.

Previous studies have suggested that calcium/calmodulin-dependent protein kinase IV (CaMKIV) functions as a positive regulator for memory formation and that age-related memory deficits are the result of dysfunctional signaling pathways mediated by cAMP response element-binding protein (CREB), the downstream transcription factor of CaMKIV. Little is known, however, about the effects of increased CaMKIV levels on the ability to form memory in adult and aged stages. We generated a transgenic mouse overexpressing CaMKIV in the forebrain and showed that the upregulation of CaMKIV led to an increase in learning-induced CREB activity, increased learning-related hippocampal potentiation, and enhanced consolidation of contextual fear and social memories. Importantly, we also observed reduced hippocampal CaMKIV expression with aging and a correlation between CaMKIV expression level and memory performance in aged mice. Genetic overexpression of CaMKIV was able to rescue associated memory deficits in aged mice. Our findings suggest that the level of CaMKIV expression correlates positively with the ability to form long-term memory and implicate the decline of CaMKIV signaling mechanisms in age-related memory deficits.

Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization.

The aim of this study was to develop a simple and sensitive method to analyze several advanced glycation end products (AGEs) simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to apply this method to the quantitation of AGEs in brown-colored foods. The developed method enabled to separate and quantitate simultaneously seven AGEs, and was applied to the determination of free AGEs contained in various kinds of soy sauce and beer. The major AGEs in soy sauce and beer were Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1). Using the developed LC-MS/MS method, recovery test on soy sauce and beer samples showed the recovery values of 85.3-103.9% for CML, 95.9-107.4% for CEL, and 69.5-123.2% for MG-H1. In particular, it is the first report that free CML, CEL, and MG-H1 were present in beer. Furthermore, long-term storage and heating process of soy sauce increased CML and MG-H1.

Effect of roasting on properties of the zinc-chelating substance in coffee brews.

ApV is a brownish polymer with zinc-chelating activity in brewed coffee. We investigated in this study the effects of roasting on the zinc-chelating, reducing, and antioxidative activities of ApV from light-, medium-, and dark-roasted coffee. We also discuss the effect on the zinc-chelating activity of adding milk to the brewed coffee. The chelating activities of ApVs were evaluated by the tetramethyl murexide method. As the intensity of roasting increased, the yield of ApV increased, and the brown color and molecular weight of ApV respectively became darker and higher. Increasing the degree of roasting also decreased the zinc-chelating activity of ApV. The reducing activities of ApVs estimated by the indophenol method were stronger than those of ascorbic acid. Both the antioxidative activity estimated by the ABTS assay and the reducing activity of ApV increased with roasting. When milk was added to instant coffee and its ApV was prepared, the zinc-chelating activity of ApV was not changed.

Detection of changes in taste of japonica and indica brown and milled rice (Oryza sativa L.) during storage using physicochemical analyses and a taste sensing system.

Changes in the taste of japonica, hybrid, and indica brown and milled rice, stored for 10 months at low (5 degrees C, 65-70% relative humidity) and room temperatures were observed by physicochemical analyses and a novel method using a taste sensing system. During storage, some properties increased or decreased while others were fairly constant. The main taste components of cooked rice such as sweetness (sucrose) and umami tastes (glutamic acid and aspartic acid) were reduced during storage, whereas glucose and fructose increased. The increase of fat acidity and consequent decrease of the pH value of the cooking solution may contribute to the off-taste of cooked stored rice. A taste sensing system with 10 lipid membrane sensors was also used to classify new and old rice samples using principal component analysis. Fresh and room temperature stored japonica and indica rice could be clearly distinguished; however, it was not possible to differentiate the samples stored at low temperature.

The identification of vitamin E homologues in medicinal plant samples using ESI(+)-LC-MS3.

The aim of this study was to elucidate the presence of vitamin E homologues in medicinal plants. To identify various homologues in the matrix of medicinal plant samples, a method for simultaneous determination was developed using ESI(+)-LC-MS3. A complete separation of each homologue was achieved within 20 min using a PFP column and an isocratic elution system of water/methanol (10:90, v/v) at a flow rate of 0.5 mL/min. The ESI-MS condition for each homologue was optimized, and the m/z value and the fragmentation pathway of each homologue were summarized. This LC-MS3 method made it possible to detect the homologues without the effect of matrix; therefore, high sensitive analysis was established, and then, the MS3 makes it possible to extract from plants with methanol only. The LC-MS3 method was applied to identify the eight vitamin E homologues in 11 medicinal plants.

Transgenic up-regulation of alpha-CaMKII in forebrain leads to increased anxiety-like behaviors and aggression.

BACKGROUND: Previous studies have demonstrated essential roles for alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) in learning, memory and long-term potentiation (LTP). However, previous studies have also shown that alpha-CaMKII (+/-) heterozygous knockout mice display a dramatic decrease in anxiety-like and fearful behaviors, and an increase in defensive aggression. These findings indicated that alpha-CaMKII is important not only for learning and memory but also for emotional behaviors. In this study, to understand the roles of alpha-CaMKII in emotional behavior, we generated transgenic mice overexpressing alpha-CaMKII in the forebrain and analyzed their behavioral phenotypes. RESULTS: We generated transgenic mice overexpressing alpha-CaMKII in the forebrain under the control of the alpha-CaMKII promoter. In contrast to alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in anxiety-like behaviors in open field, elevated zero maze, light-dark transition and social interaction tests, and a decrease in locomotor activity in their home cages and novel environments; these phenotypes were the opposite to those observed in alpha-CaMKII (+/-) heterozygous knockout mice. In addition, similarly with alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in aggression. However, in contrast to the increase in defensive aggression observed in alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in offensive aggression. CONCLUSION: Up-regulation of alpha-CaMKII expression in the forebrain leads to an increase in anxiety-like behaviors and offensive aggression. From the comparisons with previous findings, we suggest that the expression levels of alpha-CaMKII are associated with the state of emotion; the expression level of alpha-CaMKII positively correlates with the anxiety state and strongly affects aggressive behavior.

Browning of model orange juice solution: factors affecting the formation of decomposition products.

A model solution of orange juice was prepared and stored. Factors affecting browning and formation of such decomposition products as 3-hydroxy-2-pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), furfural, 5-hydroxymaltol, and 2-furoic acid were examined. Ascorbic acid (AsA) was essential for browning, which was stimulated by amino acids and citric acid, and repressed by chelators and radical scavengers (RS). 3OH2P was derived from AsA. Its formation was stimulated by sugars and repressed by citric acid, chelating agents, and RS. HMF was derived from fructose. Furfural was derived from AsA, and its formation was stimulated by sugars and chelating agents and repressed by RS. 5-hydroxymaltol and 2-furoic acid were derived from fructose and AsA respectively. We did not find any decomposition products showing the same formation pattern as the browning, but a furfural solution with added amino acids turned brown like the model orange juice solution. It might be an indicator for the browning of orange juice.

Characterization of a metal-chelating substance in coffee.

A metal-chelating substance in brewed coffee was separated and characterized by its chemical structure. This substance was a brown polymer. The contents of sugars, amino acids and phenolics in the substance were evaluated. This polymer contained small amounts of sugars and amino acids in its partial structure. After being decomposed by alkaline fusion, the decomposition products were identified by HPLC and GC-MS. Several phenolics were detected in the decomposed products. To characterize this substance, various types of model compounds were prepared by roasting chlorogenic acid, sucrose, and (or) protein with cellulose powder. Among these model compounds, the polymer-forming ability was highest in the model prepared from all four of materials, but the metal-chelating ability was the highest in the model prepared from chlorogenic acid and cellulose. These results suggest that this metal-chelating substance was a melanoidin-like polymer formed by the decomposition and polymerization of sugars, amino acids and phenolics.

Effects of peroxidase and hydrogen peroxide on the dityrosine formation and the mixing characteristics of wheat-flour dough.

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.

Antioxidative activity of a zinc-chelating substance in coffee.

Coffee brew contains a brownish zinc-chelating polymer designated ApV. ApV was prepared from the precipitate formed in a solution of instant coffee by adding ZnCl(2) and purified using ion-exchange and cellulose column chromatographies. The antioxidative activities of ApV and related compounds were evaluated in this study. The free-radical scavenging activity of ApV estimated by ABTS assay was at a similar level to that of instant coffee, while the O(2)(-) scavenging activity of ApV, which is superoxide dismutase-like activity, was lower than that of instant coffee. The hydroxyl-radical scavenging activity of ApV was higher than that of instant coffee, and the auto-oxidation of linoleic acid was more strongly inhibited by ApV than by caffeic acid.

Quality of cut lettuce treated by heat shock: prevention of enzymatic browning, repression of phenylalanine ammonia-lyase activity, and improvement on sensory evaluation during storage.

Stored cut lettuce gradually turns brown on the cut section after several days of storage, because cutting induces phenylalanine ammonia-lyase (PAL) activity, the biosynthesis of polyphenol is promoted, and the polyphenols are oxidized by polyphenol oxidase. Here, the effect of heat shock treatment at 50 degrees C for 90 s on the quality of cut lettuce during cold storage was examined. The heat shock treatment significantly repressed the induction of PAL activity and phenolics accumulation in cut lettuce during storage, and prevented the browning of cut lettuce. Ascorbic acid content was not affected by the heat shock treatment. The sensory analysis showed that the organoleptic quality of cut lettuce treated by heat shock was significantly better than that of the control cut lettuce. These results show that heat shock treatment is useful for prolonging the shelf life of cut lettuce.

Browning and decomposed products of model orange juice.

A model solution of orange juice containing sugars, ascorbic acid, and citric acid was prepared and its browning during storage was examined. The solution gradually turned brown. Ascorbic acid (AsA) most contributed to the browning. Citric acid and such amino acids as Arg and Pro promoted the browning. DTPA, a strong chelator, inhibited the browning. 3-Hydroxy-2-pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), furfural, 5-hydroxymaltol, and 2-furoic acid were identified as decomposed products in the stored solution. When 3OH2P was stored, the solution turned slightly brown. Furfural solution added with amino acids turned yellow. 3OH2P showed a positive relation with the browning of retail orange juice during storage.

Properties of chlorogenic acid quinone: relationship between browning and the formation of hydrogen peroxide from a quinone solution.

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7 x 10(3) for 325 nm and 9.7 x 10(2) for 400 nm in an acidic aqueous solution. Chlorogenic acid and H2O2 were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H2O2 spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.