Suppressive effect of Yokukansan on glutamate released from canine keratinocytes.

Background: Canine atopic dermatitis (CAD) is caused by skin barrier dysfunction due to allergen exposure. Excessive glutamate release in the skin is associated with delayed skin barrier function recovery and epidermal thickening and lichenification. Treatment with Yokukansan (YKS), a traditional Japanese medicine, reduces dermatitis severity and scratching behavior in NC/Nga mice by decreasing epidermal glutamate levels. However, the association between canine keratinocytes and glutamate and the mechanism by which YKS inhibits glutamate release from keratinocytes remains unknown. Aim: We aimed to investigate glutamate release from canine progenitor epidermal keratinocytes (CPEKs) and the inhibitory effect of YKS on this release. We also explored the underlying mechanism of YKS to enable its application in CAD treatment. Methods: Glutamate produced from CPEKs in the medium at 24 hours was measured. The measurement conditions varied in terms of cell density and YKS concentration. CPEKs were treated with a glutamate receptor antagonist (MK-801), a glutamate transporter antagonist (THA), and a glutamate dehydrogenase inhibitor (epigallocatechin gallate; EGCG), and the inhibitory effect of YKS, YKS + THA, MK-801, and EGCG on this release was determined. MK-801 and glutamate dehydrogenase inhibitor were tested alone, and THA was tested in combination with YKS. Finally, glutamine incorporated into CPEKs at 24 hours was measured using radioisotope labeling. Results: CPEKs released glutamate in a cell density-dependent manner, inhibited by YKS in a concentration-dependent manner. Moreover, YKS reduced the intracellular uptake of radioisotope-labeled glutamine in a concentration-dependent manner. No involvement of glutamate receptor antagonism or activation of glutamate transporters was found, as suggested by previous studies. In addition, EGCG could inhibit glutamate release from CPEKs. Conclusion: Our findings indicated that glutamate release from CPEKs could be effectively inhibited by YKS, suggesting the utility of YKS in maintaining skin barrier function during CAD. In addition, CPEKs are appropriate for analyzing the mechanism of YKS. However, we found that the mechanism of action of YKS differs from that reported in previous studies, suggesting that it may have had a similar effect to EGCG in this study. Further research is warranted to understand the exact mechanism and clinical efficacy in treating CAD.

Effects of the ammonium stress on photosynthesis and ammonium assimilation in submerged leaves of Ottelia cordata - an endangered aquatic plant.

Although ammonium (NH4+-N) is an important nutrient for plants, increases in soil nitrogen (N) input and atmospheric deposition have made ammonium toxicity a serious ecological problem. In this study, we explored the effects of NH4+-N stress on the ultrastructure, photosynthesis, and NH4+-N assimilation of Ottelia cordata (Wallich) Dandy, an endangered heteroblastic plant native to China. Results showed that 15 and 50 mg L-1 NH4+-N damaged leaf ultrastructure and decreased the values of maximal quantum yield (Fv/Fm), maximal fluorescence (Fm), and relative electron transport rate (rETR) in the submerged leaves of O. cordata. Furthermore, when NH4+-N was ≥ 2 mg L-1, phosphoenolpyruvate carboxylase activity (PEPC) and soluble sugar and starch contents decreased significantly. The content of dissolved oxygen in the culture water also decreased significantly. The activity of the NH4+-N assimilation enzyme glutamine synthetase (GS) significantly increased when NH4+-N was ≥ 10 mg L-1 and NADH-glutamate synthase (NADH-GOGAT) and Fd-glutamate synthase (Fd-GOGAT) increased when NH4+-N was at 50 mg L-1. However, the activity of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase (NADH-GDH) and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADPH-GDH) did not change, indicating that GS/GOGAT cycle may play an important role in NH4+-N assimilation in the submerged leaves of O. cordata. These results show that short-term exposure to a high concentration of NH4+-N is toxic to O. cordata.

Precision cut tissue slices to investigate the effects of triclosan exposure in Mytilus galloprovincialis.

Precision-cut tissue slices (PCTS) are frequently used in mammalian research, but its application in the area of aquatic toxicology is still humble. This work proposes the use of PCTS to investigate the effects of the antimicrobial triclosan (TCS) in the mussel Mytilus galloprovincialis. PCTS sectioned from the digestive gland (400 µm) were exposed to 10, 100, and 500 nM TCS for 24 h, and the expression of selected genes, together with the biomarkers, carboxylesterases (CbE) and glutathione S-transferases (GST), and the analysis of lipids in PCTS and culture medium, were used to investigate the molecular initiating events of triclosan in the digestive gland of mussels. Significant dysregulation in the expression of phenylalanine-4-hydroxylase (PAH), glutamate dehydrogenase (GDH), fatty acid synthase (FASN), and 7-dehydrocholesterol reductase (DHCR7), involved in energy, phenylalanine and lipid metabolism, were detected. The analysis of lipids evidenced significant changes in cholesteryl esters (CEs) and membrane lipids in the culture medium of exposed PCTS, suggesting dysregulation of energy and lipid metabolism that can affect lipid dynamics in mussels exposed to triclosan.

Phosphonate analog of 2-oxoglutarate regulates glutamate-glutamine homeostasis and counteracts amyloid beta induced learning and memory deficits in rats.

BACKGROUND: Metabolic alteration is a mainstream concept underlying the cognitive decline in neurodegenerative disorders including Alzheimer's disease (AD). Mitochondrial enzyme α-ketoglutarate dehydrogenase complex (α-KGDHC) seems to play a dual-edged sword role in cytotoxic insult. Here, using succinyl phosphonate (SP), a specific α-KGDHC inhibitor, we aimed to examine its potential action on AD progression. METHODS: Male Wistar rats were assigned to two separate experiments. First, they were bilaterally microinjected into the dorsal CA1 area by amyloid-beta (Aß)25-35 for four consecutive days. Seven days after the last injection, they were trained to acquire Morris Water Maze (MWM) task for three successive days when they were treated with SP after each training session. In the second experiment, SP was administered 30 min after the first Aß microinjection and behavioral tests were performed one week after the last Aß administration. The activity of glutamate dehydrogenase (GDH), and glutamine synthetase (GS), as key enzymes involved in glutamate-glutamine homeostasis and histological assays were evaluated in the hippocampi. RESULTS: Our behavioral results indicated that post-training SP treatment enhanced task acquisition but did not change memory performance in Aß-treated rats. However, administration of SP at the time of Aß injection precludes the deteriorative effect of Aß and neuronal injury on both spatial learning and memory performances indicating its preventive action against Aß pathology at its early stages. Measurement of enzymes activity shows that α-KGDHC activity was reduced in the Aß treated group, and SP administration restored its activity; also, GDH and GS activities were increased and decreased respectively due to Aß, and SP reversed the action of Aß on these enzymes. CONCLUSIONS: This study proposes that SP possibly a promising therapeutic approach to improve memory impairment in AD, especially in the early phases of this disease.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

Antidiabetic and neuroprotective effects of a novel repaglinide analog.

Repaglinide (RPG) is an oral insulin secretagogue used in the treatment of diabetes. In this study, a new RPG analog was synthesized. Its antidiabetic and neuroprotective effects on dorsal root ganglions (DRG) in streptozotocin (STZ)-induced diabetic rats were examined compared to RPG. To assess the effects of 2-methoxy-4-(2-((3-methyl-1-(2-(piperidin-1-yl)phenyl)butyl)amino)-2-oxoethoxy)benzoic acid (OXR), the impact of OXR on oxidative stress biomarkers, motor function, and the expression of the glutamate dehydrogenase 1 (GLUD1), SLC2A2/glucose transporter 2 (GLUT2), and glucokinase (GCK) genes in STZ-induced diabetic rats were assessed. DRGs were examined histologically using hemotoxylin and eosin staining. Molecular docking was used to investigate the interactions between OXR and the binding site of RPG, the ATP-sensitive potassium (KATP) channel. Following 5 weeks of treatment, OXR significantly increased the level of total antioxidant power, decreased reactive oxygen species, and lipid peroxidation in the DRGs of diabetic rats. OXR restored STZ-induced pathophysiological damages in DRG tissues. Administration of OXR improved motor function of rats with diabetic neuropathy. Administration of 0.5 mg/kg OXR reduced blood glucose while promoting insulin, mainly through upregulation of messenger RNA expression of GLUD1, GLUT2, and GCK in the pancreas. Molecular docking revealed a favorable binding mode of OXR to the KATP channel. In conclusion, OXR has neuroprotective effects in diabetic rats by lowering oxidative stress, lowering blood glucose, and stimulating insulin secretion. We report that 0.5 mg/kg OXR administration was the most effective concentration of the compound in this study. OXR may be a promising target for further research on neuroprotective antidiabetic molecules.

A two-dimensional imaging biosensor to monitor enhanced brain glutamate release stimulated by nicotine.

In this study, we have imaged and monitored real-time release of neurotransmitter glutamate from mouse brain slices stimulated by nicotine and physiological salts using a newly developed two-dimensional (2D) imaging biosensor. Nicotine, the addictive substance contained in cigarettes and other tobacco baring products, affects human body through interactions with both central and peripheral nervous system receptors and exists in various concentrations in body organs including the brain. The 2D imaging biosensor, designed for sensitive glutamate monitoring, is prepared through the use of a flat silica plate that is covalently attached with an enzyme, glutamate dehydrogenase (GDH). The 2D imaging biosensor can spatially resolve glutamate release and has a detection limit of 40 nM in detecting glutamate release from glutaminergic neurons in the brain slices. Using this biosensor, we have shown that the administration of nicotine (50 nM) on the brain slice stimulates repetitive glutamate release. To the best of our knowledge, this is the first time that a biosensor was used to define the vital role of nicotine in glutamate release. Physiological salts such as KCl and CaCl(2) have also been used to characterize the biosensor in measuring glutamate release. It is found in this study that nicotine stimulation is much more potent than physiological salts. The real-time detection of newly released glutamate from mouse brain slices clearly demonstrates the feasibility of the two-dimensional glutamate biosensor for real-time monitoring of dynamic glutamate release from living specimen.

Glutamine synthetase and glutamate dehydrogenase in the prefrontal cortex of patients with schizophrenia.

Basing primarily on the facts of altered levels of glutamate neurotransmitter, its receptors and transporters in schizophrenic brain, the "glutamatergic hypothesis" of schizophrenia has been broadened into the field of brain glutamate metabolism. Significantly changed levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the key enzymes involved in glutamine-glutamate cycling between neurons and glia, have been found in the prefrontal cortex (area 10) of patients with schizophrenia compared to controls (P<.01). The data were obtained by enzymatic activity determinations as well as immunoreactivity level evaluations for GS, glutamine synthetase-like protein (GSLP), and three GDH isoenzymes in brain extracts by immunoblotting using specific polyclonal and monoclonal antibodies. Inverse changes in amounts of proteins of GS and GSLP, as well as elevation in amounts of GDH isoenzymes have been observed in schizophrenia. The presented results provide evidence for the impairment of glutamate metabolism and, in turn, abnormalities in functioning of the glutamate-glutamine cycle in the frontal cortex of patients with schizophrenia.

Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes.

A growing body of evidence proposes that glial cells have the potential to play a role as modulators of neuronal activity and synaptic transmission by releasing the neurotransmitter glutamate (Arague et al., 1999). We explore the spatial nature of glutamate release from astrocytes with an enzyme-linked assay system and CCD imaging technology. In the presence of glutamate, L-glutamic dehydrogenase (GDH) reduces NAD(+) to NADH, a product that fluoresces when excited with UV light. Theoretically, provided that GDH and NAD(+) are present in the bathing saline, the release of glutamate from stimulated astrocytes can be optically detected by monitoring the accumulation of NADH. Indeed, stimuli that induce a wave of elevated calcium among astrocytes produced a corresponding spread of extracellular NADH fluorescence. Treatment of cultures either with thapsigargin, to deplete internal calcium stores, or with the membrane-permeant calcium chelator BAPTA AM significantly decreased the accumulation of NADH, demonstrating that this fluorometric assay effectively monitors calcium-dependent glutamate release. With a temporal resolution of 500 msec and spatial resolution of approximately 20 micrometer, discrete regions of glutamate release were not reliably resolved. The wave of glutamate release that underlies the NADH fluorescence propagated at an average speed of approximately 26 micrometer/sec, correlating with the rate of calcium wave progression (10-30 micrometer/sec), and caused a localized accumulation of glutamate in the range of 1-100 microM. Further analysis of the fluorescence accumulation clearly demonstrated that glutamate is released in a regenerative manner, with subsequent cells that are involved in the calcium wave releasing additional glutamate.

In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver.

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.

Glutamate dehydrogenase improves binding of [3H]CGP39653 to NMDA receptors in the autoradiographic assay.

The novel high-affinity competitive NMDA receptor antagonist, CGP39653, is employed as radioligand to autoradiographically label the NMDA receptor in rat and human brain. Glutamate dehydrogenase (GlDH; E.C.1.4.1.3) was added to the incubation buffer to degrade residual endogenous L-glutamate, which was not entirely removed from the section after the prewashing step and interfered with [3H]CGP39653 binding. At 20 nM [3H]CGP39653, GLDH increased specific binding as much as 5 times, depending on the dose of GlDH and the presence of NAD+, and hydrazine. Scatchard plots of binding data revealed that this increase was due to a decrease of the KD from 148 nM to 33 nM in the absence and the presence of GlDH, respectively. Addition of GlDH in the NMDA receptor autoradiographic assay may be of importance in quantitative studies with human brain tissue which may contain variable levels of endogenous L-glutamate.

Effects of in vivo perfusion of glutamate dehydrogenase in the guinea pig cochlea on the VIIIth nerve compound action potential.

Glutamate is considered as the best candidate for the neurotransmission between the inner hair cell and the primary efferent neurons in the mammalian cochlea. In order to test its presence in the synapse, a degradative enzyme for glutamate, glutamate dehydrogenase (GDH) was perfused in the cochlea of guinea pigs. The intensity function of the VIIIth nerve compound action potential was recorded as a physiological test. The results show that the GDH induces a decrease in the auditory nerve responsiveness. The threshold elevation observed is dependent upon the enzyme concentration.

Development and improvement of a commercial uric acid enzymatic determination kit on a centrifugal fast analyzer.

The performance of a uric acid determination kit has been evaluated for five months, under routine conditions, in a General Hospital Biochemical Laboratory. An anomalous increment in fresh serum blanks was noted in the kit when first introduced. This interference, probably due to alcohol dehydrogenase contamination, was corrected by addition of 50 mmol/l (NH4)2SO4 to the reagent. Results obtained with this modified reagent correlate perfectly with those obtained with modified kits by Smith Kline Instruments (SKI), and with many other determination methods. Correlations are discussed and explanations for differences in statistical data are offered. Within run and between run precision data are presented. The kit fits perfectly with the needs of centrifugal fast analyzers and discrete micro analyzers, on account of its speed, reliability and precision.

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate: production of an ammonia-containing intermediate in the "burst" phase.

We have studied the effects of ammonium acetate on the transient "burst" phase of the oxidation of L-glutamate by glutamate dehydrogenase. Two measurable changes are observed in the "burst" phase as ammonium acetate concentration is increased: (i) an increase in the apparent first-order rate constant, kapp, and (ii) a decrease in the amplitude of the absorbance change measured at 320 nm. The increase in kapp shows a hyperbolic dependence on ammonium acetate concentration and is independent of glutamate concentration. The results demonstrate the existence of an intermediate immediately following hydrogen transfer. The intermediate contains enzyme, reduced coenzyme, ammonia, and alpha-ketoglutarate moieties and is in equilibrium with the known complex consisting of enzyme, reduced coenzyme, and alpha-ketoglutarate. At high concentrations of ammonium acetate, the equilibrium favors the ammonia containing complex.

[Purification and properties of the NADP-specific glutamate dehydrogenase of Candida tropicalis feed yeasts]. / Ochistka i nekotorye svoistva NADF-spetsifichnoi glutamatdegidrogenazy kormovykh drozhzhei Candida tropicalis.

From the cell-free extract of fodder yeast Candida tropicalis NADP-specific glutamate dehydrogenase was isolated and partially purified (75-fold) by means of fractional precipitation by ammonium sulphate and ion-exchange chromatography on DEAE-cellulose. The preparation was investigated with the aid of polyacrylamide gel electrophoresis. Kinetic characteristics of the enzyme in the cell-free extract and partially purified preparation were derived.