Glutamate Dehydrogenase Activity Research Articles

The metabolic and toxic acute effects of phloretin in the rat liver

The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the FoF1–ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.

Fluopyram inhibits tricarboxylic acid cycle but induces γ-aminobutyric acid-shunt pathway to prolong postharvest shelf life of litchi fruit

Rapid pericarp browning results in a short shelf life of postharvest litchi fruit. In this study, the impact of fluopyram (Flu) on litchi pericarp browning and possible mechanisms were investigated. Results showed that application of 0.2 % (v/v) fluopyram (Flu) delayed litchi pericarp browning during storage at 25 °C. Respiration rate and tricarboxylic acid (TCA) cycle were reduced in Flu-treated fruit, as indicated by the decreased succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (α-KGDH) activities. Meanwhile, Flu treatment resulted in higher succinic acid content but lower fumaric acid and α-ketoglutarate contents compared to those in non-Fu-treated fruit. Furthermore, Flu pretreatment maintained higher glutamate (Glu) level via enhancing glutamate dehydrogenase (GDH) activity. Additionally, Flu treatment accelerated gamma-aminobutyric acid (GABA)-shunt pathway by regulating GABA content and activities of glutamate decarboxylase (GAD) as well as GABA transaminase (GABA-T). Importantly, higher ATP and GTP contents were observed in Flu-treated fruit. Taken together, these results suggested that Flu treatment inhibited respiration but accelerated GABA-shunt pathway, which might contribute to higher energy status and thus attenuate litchi pericarp browning. This study provided new insights of Flu application in quality maintenance and shelf life extension of postharvest fruit storage.

Synchronised interaction of carbon and nitrogen provides drought tolerance in Cyamopsis tetragonoloba

Drought is a major adverse environmental factor that limits plant productivity. Concomitant assimilation of carbon (C) and nitrogen (N) in illuminated leaves requires the regulated partitioning of reductant and photosynthate to sustain the demands of amino acid and carbohydrate biosynthesis. The short-term exposure to drought and its responses of photosynthesis and photosynthate partitioning to N enrichment was studied in Cyamopsis tetragonoloba (L.) Taub (guar). Four contrasting varieties of guar, HG-563, RGC-986 (drought-tolerant) and RGC-471, Varsha (drought-sensitive) were subjected to 15 days water stress and leaf tissues were studied in order to contemplate the counter strategy engaged under drought conditions. In guar plants, sensors trigger a signalling cascade that activates numerous proteins to induce the expression of certain genes involved in carbon and nitrogen metabolism.The levels of glucose, starch, and Glutamate dehydrogenase (GDH), Aspartate aminotransferase (AspAT), isocitrate dehydrogenase (ICDH), Nitrate Reductase (NR), and Nitrite reductase (NiR) activities were determined biochemically during 15 days drought treatment, at three stages. Further, the carbon (oxidative phosphorylation and TCA cycle) - nitrogen metabolic pathway interaction was studied at the molecular level. The respiratory pathway genes were upregulated, while the nitrogen metabolic pathway genes were downregulated under all stages of drought in guar plants. The physiological, biochemical and molecular data suggests that the high rates of dark respiration was insufficient to provide extra carbon required to assist amino acid synthesis but rather sufficed by a rapid metamorphosis in the photosynthetic carbon partitioning to amino acid metabolism via the nitrogen metabolism. • Drought causes NADH DEHYDROGENASE gene to escalate dark respiration. • Nitrogen assimilation reduces under drought in leguminous Cyamopsis tetragonoloba. • Drought triggers protein kinase cascade signalling in guar plants. • Drought regulates nitrogen metabolic genes in guar. • Drought triggers increase in sugar content at the cost of amino acids.

Nitrogen and Chemical Control Management Improve Yield and Quality in High-Density Planting of Maize by Promoting Root-Bleeding Sap and Nutrient Absorption.

High-density planting aggravates competition among plants and has a negative impact on plant growth and productivity. Nitrogen application and chemical control can improve plant growth and increase grain yield in high-density planting. Our experiment explored the effects of nitrogen fertilizer and plant growth regulators on maize root-bleeding sap, phosphorus (P) and potassium (K) accumulation and translocation, and grain yield and quality in high-density planting. We established a field study during the 2017 and 2018 growing seasons, with three nitrogen levels of N100 (100 kg ha−1), N200 (200 kg ha−1), and N300 (300 kg ha−1) at high-density planting (90,000 plants ha−1), and applied Yuhuangjin (a plant growth regulator mixture of 3% DTA-6 and 27% ethephon) at the 7th leaf. Our results showed that N200 application combined with chemical control could regulate amino acid and mineral nutrient concentration delivery rates in root-bleeding sap and improve its sap rate. Also, the treated plant exhibited higher P and K uptake and translocation ability. Furthermore, chemical control and N200 treatment maintained a high level of ribulose-1,5-bisphosphate carboxylase (RuBPCase), phosphoenolpyruvate carboxylase (PEPCase), nitrate reductase (NR), and glutamine synthetase (GS) enzymatic activities in leaves. In addition, plant growth regulator and nitrogen application improved the enzymatic activities of GS, glutamate dehydrogenase (GDH), and glutamic pyruvic transaminase (GPT) and the contents of crude protein, lysine, sucrose, and soluble sugar in grain and ultimately increased maize yield. This study suggests that N200 application in combination with chemical control promotes root vitality and nutrient accumulation and could improve grain yield and quality in high-density planting.

Alterations in Inflammatory Cytokines and Redox Homeostasis in LPS-Induced Pancreatic Beta-Cell Toxicity and Mitochondrial Stress: Protection by Azadirachtin

Inflammation and redox imbalance are hallmarks of cancer, diabetes, and other degenerative disorders. Pathophysiological response to these disorders leads to oxidative stress and mitochondrial dysfunction by alterations and reprogramming in cellular signaling and metabolism. Pancreatic beta cells are very sensitive to the inflammatory and altered nutrient signals and hence play a crucial role in diabetes and cancer. In this study, we treated insulin-secreting pancreatic beta cells, Rin-5F, with the bacterial endotoxin, LPS (1 μg/ml) to induce an inflammatory response in vitro and then treated the cells with a known anti-inflammatory, anticancer and antioxidant phytochemical, azadirachtin (AZD, 25 µM for 24 h). Our results demonstrated lipid peroxidation and nitric oxide production causing increased nitro/oxidative stress and alterations in the activities of anti-oxidant enzymes, superoxide dismutase and catalase after LPS treatment. Pro-inflammatory responses caused by translocation of nuclear factor kappa B and release of inflammatory cytokines were also observed. These changes were accompanied by GSH-dependent redox imbalance and alterations in mitochondrial membrane potential and respiratory complexes enzyme activities leading to mitochondrial respiratory dysfunction, reduced ATP synthesis, and intrinsic caspase-9 mediated apoptosis. Caspase-9 was activated due to alterations in Bcl-2 and Bax proteins and release of cytochrome c into the cytosol. The activities of oxidative stress-sensitive mitochondrial matrix enzymes, aconitase, and glutamate dehydrogenase were also inhibited. Treatment with AZD showed beneficial effects on the recovery of antioxidant enzymes, inflammatory responses, and mitochondrial functions. GSH-dependent redox homeostasis also recovered after the treatment with AZD. This study may help in better understanding the etiology and pathogenesis of inflammation-induced disorders in pancreatic beta cells to better manage therapeutic strategies.

The modulatory effects of alkaloid extracts of Cannabis sativa, Datura stramonium, Nicotiana tabacum and male Carica papaya on neurotransmitter, neurotrophic and neuroinflammatory systems linked to anxiety and depression.

This study investigated the modulatory effects of alkaloid extracts of Cannabis sativa (CSAE), Datura stramonium (DSAE), Nicotiana tabacum (NTAE) and male Carica papaya (CMAE) on neurotransmitter, neurotrophic and neuro-inflammatory systems linked to anxiety and depression. Male Wistar rats were orally administered the alkaloid extracts in doses of 5, 50, 500, and 2000mg/kg for 90days. On day 91, neurobehavioural studies were evaluated, rats were sacrificed, brain hippocampus removed and tissue homogenate prepared. Biochemical, cytokine and neurotransmitter metabolisms were estimated in the hippocampus. Expressions of genes linked to anxiety and depression were evaluated by RT-qPCR. Results showed CSAE, NTAE and CMAE act as anxiolytic and antidepressant agents by depleting TNF-α, IL-1β and reactive oxygen species concentrations, and monoamine oxidase, angiotensin 1-converting enzyme and acetylcholinesterase activities while elevating IL-10 and dopamine concentrations and glutamate dehydrogenase activity at doses of 5, 50 and 500. Same doses of CSAE, NTAE and CMAE also depleted the gene expressions of GSK3β, JNK, NF-ĸB, and Nesfatin-1 while increasing expressions of CREB, BDNF, serotonin and Nrf2. However, administration of DSAE and 2000mg/kg CSAE, NTAE and CMAE had adverse modulatory effects on the neurochemical concentrations and activities as well as the gene expressions of the evaluated neurotransmitter, neurotrophic and inflammatory systems. In conclusion, the study established the sub-chronic instrumentalization potential of CSAE, CMAE, and NTAE for anxiolytic and anti-depressive moods, though their use may be associated with dependence and addiction, which may result in more detrimental effects than any therapeutic potential they may proffer.

Late onset schizophrenia and delusional disorder: activity of platelet energy, glutamate, and glutathione metabolizing enzymes

IntroductionAlterations of glutamate, energy and glutathione metabolism contribute to the pathogenesis of psychotic disordersObjectivesRevealing clinical-biological correlations in patients with late onset schizophrenia and delusional disorder by determining clinical parameters and activity of platelet enzymes of energy, glutamate, and glutathione metabolism.Methods27 women of 45-80 years old were studied, with late onset schizophrenia or delusional disorder. Activity of platelet cytochrome c-oxidase (COX), glutamate dehydrogenase (GDH), glutathione reductase (GR) and glutathione-S-transferase (GST), and scores by PANSS, HAMD, MMSE, and CGI-S were evaluated twice: before and after the 28-th day of treatment. Activity of COX, GDH, GR, and GST was measured once in 23 women of 44-81 years old comprising the control group.ResultsAs compared with controls, only GDH activity was found significantly decreased (before and after treatment, p<0.001). Clusterisation of patients by enzymatic activities resulted in 3 clusters significantly different by COX, GDH and GST. Significant correlations were found between enzymatic activities and scores by psychometric scales: in the cluster 1 (n=9) baseline COX activity correlated with scores by PANSS positive subscale (R=0.9, p=0.001) and with scores by MMSE (R=-0.9, p=0.002); in the cluster 2 (n=12) GR activity after treatment negatively correlated with scores by PANSS (R=-0.9, p=0.001), PANSS negative subscale (R=-0.8, p=0.004), and CGI-S (R=-0.9, p=0.001).ConclusionsThe revealed correlations between enzymatic activities and clinical parameters give hope on detection of useful biochemical markers which, after enlargement of patients’ group with late onset psychotic disorders, would be validated for prediction of the pharmacotherapy efficiency and outcome of treatment.DisclosureNo significant relationships.

Nickel Toxicity Interferes with NO3-/NH4+ Uptake and Nitrogen Metabolic Enzyme Activity in Rice (Oryza sativa L.).

The excessive use of nickel (Ni) in manufacturing and various industries has made Ni a serious pollutant in the past few decades. As a micronutrient, Ni is crucial for plant growth at low concentrations, but at higher concentrations, it can hamper growth. We evaluated the effects of Ni concentrations on nitrate (NO3−) and ammonium (NH4+) concentrations, and nitrogen metabolism enzyme activity in rice seedlings grown in hydroponic systems, using different Ni concentrations. A Ni concentration of 200 μM significantly decreased the NO3− concentration in rice leaves, as well as the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthetase (GOGAT), respectively, when compared to the control. By contrast, the NH4+ concentration and glutamate dehydrogenase (GDH) activity both increased markedly by 48% and 46%, respectively, compared with the control. Furthermore, the activity of most active aminotransferases, including glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), was inhibited by 48% and 36%, respectively, in comparison with the control. The results indicate that Ni toxicity causes the enzymes involved in N assimilation to desynchronize, ultimately negatively impacting the overall plant growth.

β Cell-specific deletion of Zfp148 improves nutrient-stimulated β cell Ca2+ responses.

Insulin secretion from pancreatic β cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that β cell–specific deletion of Zfp148 (β-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from β‑Zfp148KO and control mice fed both a chow and a Western-style diet. β-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. β-Zfp148KO islets also exhibited elevated sensitivity to amino acid–induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of β-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, β-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving β cell function that are robust to the metabolic challenge imposed by a Western diet.

Changes in fermentation profile of the reticulorumen and hindgut, and nutrient digestion in dry cows fed concentrate-rich diets supplemented with a phytogenic feed additive

This study evaluated the effects of duration of high-concentrate feeding on ruminal and fecal fermentation profile, as well as selected systemic health biomarkers in nonlactating cows supplemented with or without a phytogenic feed additive (PHY). In addition, ruminal degradation kinetics and total-tract nutrient digestibility were evaluated when feeding either only forage or a high-concentrate diet. Nine nonlactating, cannulated Holstein cows were used in a crossover design. Each period included 1 wk of forage feeding (wk 0), diet transition, and 4 wk on the high-concentrate diet (1, 2, 3 and wk 4; 65% dry matter basis). Cows received PHY or not (control). Compared with wk 0, from wk 1 onward, cows on high concentrate showed greater reticular, ruminal, and fecal total volatile fatty acids (VFA), with a greater level of VFA in the rumen than in the hindgut. However, ruminal fermentation was modulated differently by PHY, which showed increased total VFA in wk 1 and increased butyrate in wk 2 in the particle-associated fluid of rumen. In the hindgut, PHY increased propionate in wk 3. Cows fed a high-concentrate diet from wk 1 and onward also showed greater ruminal lactate, as well as lower ruminal and fecal pH, independent of PHY. In addition, compared with cows in wk 1 on a high-concentrate diet, cows in wk 4 had a greater total VFA in free fluid of the rumen and lower fecal pH. Compared with cows at wk 0, cows at wk 1 on high concentrate onward showed greater serum amyloid A and greater activity of glutamate dehydrogenase. In contrast, the high-concentrate diet decreased in situ ruminal degradability of grass silage but increased degradability of corn grain as well as total-tract nutrient digestibility, with total-tract neutral detergent fiber digestibility being greater for cows on the PHY treatment. Overall, from the start of high-concentrate feeding, gut fermentation increased, but differently according to location or PHY, with a stronger build-up of VFA in the rumen compared with the hindgut. In addition, a longer duration on high concentrate exacerbated gut acidification. The enhancing effects of PHY on total VFA and butyrate in particle-associated fluid of the rumen suggest beneficial effects of PHY on particle-associated bacteria, likely contributing to the increased neutral detergent fiber digestibility. The greater production of ruminal butyrate with PHY may be beneficial for the host, given the health benefits of this acid, but more research is needed to elucidate the effects on gut microbiota and the effects of increased butyrate in nonlactating dairy cows.

Effect of pH Dependent Substrate Inhibition on Glutamate Dehydrogenase Kinetics

Glutamate dehydrogenase (GDH) serves as the main regulated enzyme linking protein and carbohydrate metabolism. This enzyme converts the amino acid glutamate to the TCA intermediate, α‐ketoglutarate. While dysregulation of GDH is linked to hyperinsulinism and other diseases, the kinetics this enzyme of have only been analyzed under dilution conditions. However, cells are more crowded than these buffer solutions frequently used to collect enzyme kinetics data because their cytoplasm contains high concentrations of proteins, carbohydrates, and other macromolecules. Evidence has shown that this macromolecular crowding slow diffusion rates and changes protein conformations, which alters the kinetics of enzymes. Scientists often mimic the crowded environment of a cell using large, synthetic polymers, such as dextran, a glucose polymer, which excludes volume to other biological molecules in the same solution. While several studies have characterized these excluded volume effects on enzyme kinetics, this is the first study to analyze the effects of crowding on enzyme inhibition. Investigating how inhibitors like GTP and activators like leucine effect the kinetics of GDH will provide a deeper understanding of the regulation of this enzyme inside out bodies. Furthermore, saturating amounts of glutamate results in substrate inhibition of GDH by forming a “dead end complex” comprised of glutamate, NADH, and GDH at low pH values. To understand the effects of crowding on substrate inhibition, GDH Michaelis Menten kinetics were analyzed in the presence and absence of dextran at four pH values. These assays were repeated with glucose, the monomer of dextran as a control to separate chemical effects from excluded volume effects caused by the large size of dextran. The presence of dextran resulted in more substrate inhibition than in the presence of glucose and this inhibition that was observed at higher pH values than in buffer alone. Leucine, a known activator of GDH, operates by destabilizing the dead‐end complex. While the presence of leucine decreases substrate inhibition in dilute conditions, this compound was unable to active GDH in the presence of dextran. Overall, these results suggest that crowding promotes substrate inhibition by stabilizing the dead‐end complex of GDH which is a stronger interaction than leucine dissociation of the dead‐end complex. Therefore, macromolecular crowding in a cell could provide an additional level of regulation on GDH inside cells.

The effects of fullerene on photosynthetic apparatus, chloroplast-encoded gene expression, and nitrogen assimilation in Zea mays under cobalt stress.

Carbon nanostructures, such as the water-soluble fullerene (FLN) derivatives, are considered perspective agents for agriculture. FLN can be a novel nano-agent modulating plant response against stress conditions. However, the mechanism underlying the impacts of FLN on plants in agroecosystems remains unclear. Zea mays was exposed to exogenous C60 -FLN applications (FLN1: 100; FLN2: 250; and FLN3: 500 mg L-1 ) with/without cobalt stress (Co, 300 μM) for 3 days (d). In the maize chloroplasts, Co stress disrupted the photosynthetic efficiency and the expression of genes related to the photosystems (psaA and psbA). FLNs effectively improved the efficiency and photochemical reaction of photosystems. Co stress induced the accumulation of reactive oxygen species (ROS) as confirmed by ROS-specific fluorescence in guard cells. Co stress increased only chloroplastic superoxide dismutase (SOD) and peroxidase (POX). Stress triggered oxidative damages in maize chloroplasts, measured as an increase in TBARS content. In Co-stressed seedlings exposed to FLN1 and FLN2 exposures, the hydrogen peroxide (H2 O2 ) was scavenged through the nonenzymes/enzymes-related to the AsA-GSH cycle by preserving ascorbate (AsA) conversion, as well as GSH/GSSG and glutathione (GSH) redox state. Also, the alleviation effect of FLN3 against stress could be attributed to increased glutathione S-transferase (GST) activity and AsA regeneration. FLN applications reversed the inhibitory effects of Co stress on nitrogen assimilation. In maize chloroplasts, FLN increased the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH), nitrite reductase (NiR), and glutamine synthetase (GS), which provided conversion of inorganic nitrogen (N) into organic N. The ammonium (NH4 + ) toxicity was removed via GS and GDH but not glutamate synthase (GOGAT). The increased NAD-GDH (deaminating) and NADH-GDH (aminating) activities indicated that GDH was needed more for NH4 + detoxification. Therefore, FLN exposure to Co-stressed maize plants might play a role in N metabolism regarding the partitioning of N assimilates. Exogenous FLN conceivably removed Co toxicity by improving the expressions of genes related to reaction center proteins of photosystems, increasing the level of enzymes related to the defense system, and improving the N assimilation in maize chloroplasts.

Macromolecular Crowding Slows Glutamate Dehydrogenase Kinetics

Most of our understanding of enzyme kinetics is based on experiments conducted in dilute solution, which does not accurately represent crowded cellular environments. Cells contain high concentrations (300‐400g/L) of various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume of solvent available for other molecules in the solution and has previously been shown to impact the behavior of enzymes. To study these potential consequences, the Michaelis‐Menten kinetics of glutamate dehydrogenase (GDH) was monitored in the presence of high concentrations of “crowding agents” such as dextran, a glucose polymer. Given this enzyme’s central role at interface of nitrogen and carbohydrate metabolism, GDH is highly regulated. Our results suggest that crowding may influence this regulation. For example, leucine is a less effective activator of GDH in the presence of dextran. Furthermore, crowding enhances substrate inhibition of GDH in a pH‐dependent manner. The resulting effects from crowding were substrate dependent such that the presence of dextran decreased the Vmax more with the NADP+ cofactor than with NAD+. Both dextran and BSA decreased the Km of NADP+ but did not affect NAD+. The effects of crowding on the enthalpy (ΔH҂) and entropy (ΔS҂) of activations determined from Eyring plots also were also substrate and pH‐dependent. Overall, these results highlight the importance of studying enzymes especially heavily regulated ones like GDH under crowded conditions that more realistically mimic the crowded cellular environment.

Effect of S-Nitrosylation of RIP3 Induced by Cerebral Ischemia on its Downstream Signaling Pathway

ObjectivesOur preliminary experiments indicate that receptor-interacting protein 3 (RIP3) is S-nitrosylated and contributes to its autophosphorylation (activation) after 3 h of rat brain ischemia/reperfusion mediated by activation of the N-methyl-D-aspartate receptor (NMDAR)-dependent neuronal NO synthase (nNOS) and is involved in the process of neuronal injury. Here, we will to demonstrate whether S-nitrosylation of RIP3 facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death. Materials and methodsAdult male Sprague-Dawley rat transient brain ischemia/reperfusion and cortical neurons oxygen and glucose deprivation (OGD)/reoxygenation models were performed. The hippocampal CA1 regions or cultured cells were homogenized and the cytosolic fraction were collected as tissue samples. Coimmunoprecipitation and western blot analysis were carried out for detecting phosphorylation of RIP1 and mixed lineage kinase-like domains (MLKL) and the Cleaved-Caspase8 (Cl-Caspase8). The activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1) were detected with ultraviolet absorption method. ResultsThis study showed that active RIP3 could phosphorylate RIP1 and MLKL through its kinase activity, promote the conversion of Caspase8 to active Cl-Caspase8, enhance the activities of PYGL, GLUL and GLUD1, and finally aggravate neuronal injury in cerebral ischemia/reperfusion. The inhibition of RIP3 S-nitrosylation inhibited the phosphorylation of RIP1 and MLKL, inhibited the activities of Caspase8, PYGL, GLUL, and GLUD1, and alleviated neuronal damage in cerebral ischemia/reperfusion. ConclusionsS-nitrosylation of RIP3 increased RIP1 and MLKL phosphorylation levels, Cl-Caspase8 content and PYGL, GLUL and GLUD1 activities and aggravated neuronal damage during cerebral ischemia/reperfusion and regulating the S-nitrosylation of RIP3 and its downstream signaling pathway might be a therapeutic target for stroke.

Effects of Parity and Stage of Lactation on Trend and Variability of Metabolic Markers in Dairy Cows

Simple SummaryThe rise in milk yield per cow, herd size, and the percentage of primiparous cows in dairy herds increasingly requires optimized health management in order to ensure the health of the cows. The transition period (three weeks before to three weeks after calving) has a key role in health problems, because dairy cows undergo tremendous metabolic changes. Metabolic monitoring provides an in-depth insight into how the cows cope with these challenges. A remarkable variability in the metabolic parameters reflects the adaptation of dairy cows during the transition from pregnancy to lactation. In addition, primiparous cows undergo physical adaptations because of growth, first gestation, the maturation of the mammary glands, the onset of lactation, and fighting for social dominance. Previous studies have rarely considered these specific demands due to the influences of parity and the lactation stage. Thus, the objective of our study was to describe the variation in metabolic parameters due to parity and the stage of lactation based on a huge number of primiparous and multiparous cows, observed at all stages of lactation, in a retrospective analysis of laboratory data. The remarkable impact of both parity and lactation was elucidated for most parameters. This should be taken into account for a correct interpretation of the laboratory diagnostics in the framework of metabolic monitoring.Metabolic monitoring is a tool that is helpful with the increasing requirements regarding feeding and health management in dairy herds. This study aimed at describing the trend and variability of different biochemical parameters in blood and urine in relation to the stages of lactation and parity, in a retrospective analysis of laboratory data from clinically healthy German Holstein cows. The results were derived from metabolic monitoring in Thuringia (Germany), during 2009–2019. A total of 361,584 measured values, of 13 different metabolic variables, were assigned to parity (primiparous and multiparous) and stage of lactation (10 classes from −30 to 300 days in milk). The Kruskal–Wallis test was applied for the evaluation of differences regarding parity or the stage of lactation. Non-esterified fatty acids, beta hydroxybutyrate, and the activity of aspartate aminotransferase in serum were clearly affected by parity and lactation. Serum concentrations of cholesterol, bilirubin, and phosphorus, as well as the serum activity of glutamate dehydrogenase, were affected by the stage of lactation, while parity impacted urea concentration. The serum activity of creatine kinase, serum concentrations of calcium, and urine concentrations of net acid base excretion, potassium, and sodium were not affected by parity or lactation. In conclusion, specific reference limits, with respect to parity and the stage of lactation, are necessary.

Ammonium removal by erythrocyte-bioreactors based on glutamate dehydrogenase from Proteus sp. jointly with porcine heart alanine aminotransferase

Excessive ammonium blood concentration causes many serious neurological complications. The medications currently used are not very effective. To remove ammonium from the blood, erythrocyte-bioreactors containing enzymes that processing ammonium have been proposed. The most promising bioreactor contained co-encapsulated glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT). However, a low encapsulation of a commonly used bovine liver GDH (due to high aggregation), makes clinical use of such bioreactors impossible. In this study, new bioreactors containing ALT and non-aggregating GDH at higher loading were first produced using the flow dialysis method and the new bacterial GDH enzyme from Proteus sp. The efficacy of these erythrocyte-bioreactors and their properties (hemolysis, osmotic fragility, intracellular and extracellular activity of included enzymes, erythrocyte indices, and filterability) were studied and compared with native cells during 1-week storage. The ammonium removal rate in vitro by such erythrocyte-bioreactors increased linearly with an increase in encapsulated GDH activity. Alanine in vitro increased in accordance with ammonium consumption, which indicated the joint functioning of both included enzymes. Thus, novel bioreactors for ammonium removal containing GDH from Proteus sp. are promising for clinical use, since they have a more efficient GDH encapsulation and their properties are not inferior to previously obtained erythrocyte-bioreactors.

Rhizobium Symbiosis Leads to Increased Drought Tolerance in Chinese Milk Vetch (Astragalus sinicus L.)

Chinese milk vetch (Astragalus sinicus L.) is an important leguminous green manure that frequently suffers from seasonal drought. To improve its drought tolerance, the effects of rhizobia inoculation on drought tolerance and the underlying physiological mechanism were investigated. Drought tolerance in combination with nitrogen assimilation, free amino acids, and polyamines was investigated in milk vetch with active nodules (AN), with inactive nodules (LN), or without nodules (NN). AN plants had increased drought tolerance compared to LN and NN plants. Glutamine synthetase (GS), glutamine 2-oxoglutarate amino transferase (GOGAT), and glutamate dehydrogenase (GDH) activities were decreased after drought, with higher levels in AN plants than in LN and NN plants under both control and drought stress conditions. Higher levels of proline (Pro), arginine (Ala), alanine (Ala), and glutamate (Glu) were observed in AN plants compared with LN and NN plants. Putrescine (Put), spermidine (Spd), and spermine (Spm) levels were increased in response to drought, and higher levels of Put and Spd were maintained in AN plants. It is suggested that active nodulation leads to increased drought tolerance in milk vetch, which is associated with improved nitrogen fixation and ammonium assimilation, which in turn lead to the promotion of free amino-acid and polyamine synthesis.

Links of platelet glutamate and glutathione metabolism with attenuated positive and negative symptoms in depressed patients at clinical high risk for psychosis.

Aim of the study is to reveal clinical and biological correlations in patients with adolescent depression and attenuated psychotic symptoms. Activity of platelet enzymes involved in glutamate-, glutathione- and energy metabolism was evaluated in control group and in the patients, because these systems are suspected as related to pathogenesis of psychosis. Adolescents (78 men, 16-25years old) hospitalized with the first acute depressive state composed two groups: with prevalence of attenuated psychotic positive or negative symptoms (Gr1 and Gr2, 48 and 30 patients, respectively). Control group comprised 20 mentally healthy men of 19-25years old. Gr1 differed significantly from Gr2 in scores by the Scale of Prodromal Symptoms (SOPS) for positive symptoms, p < 0.001, for disorganization symptoms, p < 0.003, and for total SOPS score, p < 0.001, before the treatment started. When patients from either Gr1 or Gr2 were compared with the control group, significantly decreased baseline activities of platelet glutamate dehydrogenase (GDH), glutathione reductase (GR) and glutathione S-transferase (GST) were found (p < 0.0001). Different correlations were found between baseline enzymatic activities in Gr1 and Gr2: GDH activity correlated with GR activity in Gr1 (R = 0.37), and with GST activity in Gr2 (R = 0.70). Significant correlations were found only in Gr2 between the delta of scores by SOPS negative symptoms (SOPS-N) under treatment and baseline GDH, GST, and GR activities (R = - 0.36, R = - 0.60, and R = 0.38, respectively). The found correlations of the baseline enzymatic activity levels with the value of the decrease (delta) in SOPS-N scores under the treatment represent interest for the prediction of the pharmacotherapy efficiency.

Polyamines Metabolism Interacts with γ-Aminobutyric Acid, Proline and Nitrogen Metabolisms to Affect Drought Tolerance of Creeping Bentgrass.

Due to increased global warming and climate change, drought has become a serious threat to horticultural crop cultivation and management. The purpose of this study was to investigate the effect of spermine (Spm) pretreatment on metabolic alterations of polyamine (PAs), γ-aminobutyric acid (GABA), proline (Pro), and nitrogen associated with drought tolerance in creeping bentgrass (Agrostis stolonifera). The results showed that drought tolerance of creeping bentgrass could be significantly improved by the Spm pretreatment, as demonstrated by the maintenance of less chlorophyll loss and higher photosynthesis, gas exchange, water use efficiency, and cell membrane stability. The Spm pretreatment further increased drought-induced accumulation of endogenous PAs, putrescine, spermidine, and Spm, and also enhanced PAs metabolism through improving arginine decarboxylases, ornithine decarboxylase, S-adenosylmethionine decarboxylase, and polyamine oxidase activities during drought stress. In addition, the Spm application not only significantly improved endogenous GABA content, glutamate content, activities of glutamate decarboxylase and α-ketoglutarase, but also alleviated decline in nitrite nitrogen content, nitrate reductase, glutamine synthetase, glutamate synthetase, and GABA aminotransferase activities under drought stress. The Spm-pretreated creeping bentgrass exhibited significantly lower ammonia nitrogen content and nitrite reductase activity as well as higher glutamate dehydrogenase activity than non-pretreated plants in response to drought stress. These results indicated beneficial roles of the Spm on regulating GABA and nitrogen metabolism contributing towards better maintenance of Tricarboxylic acid (TCA) cycle in creeping bentgrass. Interestingly, the Spm-enhanced Pro metabolism rather than more Pro accumulation could be the key regulatory mechanism for drought tolerance in creeping bentgrass. Current findings provide a comprehensive understanding of PAs interaction with other metabolic pathways to regulate drought tolerance in grass species.

Longitudinal study of the effect of sporidesmin toxicity on lamb production and serum biochemistry in a flock of 46 Romney ewes using a standardised measure of liver damage

ABSTRACT Aims To evaluate the effect of sporidesmin toxicity on production outcomes and serum biochemistry analytes in mixed age Romney ewes, using a standardised measure of liver damage. Methods This was a prospective longitudinal study following 46 mixed age Romney ewes from sporidesmin intoxication in April 2019, to slaughter 8 months later. The ewes were blood-sampled up to eight times, with a panel of serum biochemistry tests performed on the final six samples. However, only gamma-glutamyl transferase (GGT) activity was measured in the first two samples collected at the end of sporidesmin intoxication and 2 weeks later. Body condition score, ewe weight and production data were also recorded. Using a standardised liver score, based on histology of liver samples collected at slaughter, ewes were assigned to one of three liver disease categories (LDC); low, middle, and high. These were then used as the outcome or predictor variables for statistical analyses. Finally, two separate decision tree models, using recursive partitioning (RP), were fitted to the biochemistry data and to the GGT data collected at FE outbreak, to predict ewes in the low LDC. Results There was no evidence of a difference for the effect of LDC on ewe weight (p = 0.86) with ewes, on average, gaining weight to weaning. Weaning percent, lamb rearing percent and ewe flock efficiency were lower in ewes with high LDC, and scanning-to-weaning lamb loss was significantly higher in sheep with high LDC (p = 0.02). Serum activities of GGT and glutamate dehydrogenase and concentration of globulin were significantly lower in sheep with low LDC than in sheep with middle or high LDC (p < 0.05). However, there was no evidence of a difference for the effect of LDC on other biochemistry variables (p > 0.05). The final RP model for the biochemistry data categorised ewes as low LDC if their GGT was <122 IU/L, 3 months after sporidesmin intoxication, or if their GGT was <514 IU/L, <18 days after sporidesmin intoxication. Conclusions and clinical relevance Sheep with gross and histological evidence of severe sporidesmin-induced liver damage were able to maintain or gain body weight, suggesting that sporidesmin intoxication alone is not causative of poor body condition. Similarly, many of the serum biochemistry tests were not associated with evidence of liver damage. Lamb production was reduced in ewes with evidence of severe liver damage and the decision tree model showed promise as a basis to select ewes for culling.