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Background and Aims: Possible protective effects of saffron (Crocus sativus L) have been reported in several randomized clinical trials (RCTs). Current systematic review was performed to summarize the efficacy of saffron intake on liver enzymes., Methods: An electronic database search was conducted on PubMed/Medline, Scopus, Web of Science, and Cochrane for RCTs comparing effect of saffron and placebo on liver enzymes from inception to July 2021. There was no restriction in language of included studies and we calculated the standardized mean difference (SMD) and 95% Confidence Intervals (CI) for each variable. Random-effect model was used to calculate effect size., Results: Eight studies (n = 463 participants) were included in the systematic review. The saffron intake was associated with a statistically significant decrease in aspartate aminotransferase (AST) (SMD: -0.18; 95% CI: -0.34, -0.02; I 2  = 0%) in comparison to placebo intake. Our results also indicated that saffron consumption did not have a significant effect on alanine aminotransferase (ALT) (SMD: -0.14; 95% CI: -0.36, 0.09; I 2  = 47.0%) and alkaline phosphatase (ALP) levels (SMD: 0.14; 95% CI: -0.18, 0.46; I 2  = 42.9%) compared to placebo., Conclusions: Saffron intake showed beneficial impacts on circulating AST levels. However, larger well-designed RCTs are still needed to clarify the effect of saffron intake on these and other liver enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Objective: We aimed to investigate the effect of switching from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF) on the hepatic safety and metabolic profile., Methods: Consecutive HIV patients, enrolled in the Surveillance Cohort Long-term Toxicity Antiretrovirals/Antivirals (SCOLTA) project, switching from TDF to TAF were included. Changes from baseline (T0) to 6-month follow-up (T1) were evaluated using paired t -test and signed rank test., Results: A total of 190 patients switched from TDF to TAF and had one 6-month follow-up visit. They were 80% male, 74.2% at CDC stage A-B, 93.7% with undetectable HIV-viral load. Mean age was 46.7±10.7 years, body mass index was 25.0±3.9 kg/m 2 , median CD4 cell count was 634 cell/µL (interquartile range [IQR]=439-900), aspartate aminotransferase (AST) was 23 (IQR=19-30) IU/L, and alanine aminotransferase (ALT) was 24 (IQR=17-34) IU/L. At T1, both AST (median=-1, IQR=-5-2 IU/L, P =0.004) and ALT (median=-2, IQR=-7-3 IU/L, P =0.0004) showed a significant decrease. Among 28 patients with ALT >40 at baseline, reduction was significant both clinically (-17, IQR=-32--1) and statistically ( P =0.0003). Total cholesterol levels (TC) increased (+13.4±3.8 mg/dL, P =0.0006), as well as HDL-cholesterol (HDL-C) (+3.8±1.2 mg/dL, P =0.02), LDL Cholesterol (LDL-C) (+7.6±3.4, P =0.03) and glucose (+4.0±1.8 mg/dL, P =0.02). D:A:D: and Framingham risk score did not change at 6 months after switch., Conclusion: A significant reduction of liver enzymes was observed after switching from TDF to TAF, especially in subjects with initial level of ALT >40 IU/L. Glucose, TC, HDL-C, and LDL-C increased, with no effect on cardiovascular risk scores., Competing Interests: Nicola Squillace reports personal fees from ViiV Healthcare and grants from Gilead science, outside the submitted work. Benedetto Maurizio Celesia reports personal fees and non-financial support from MSD, Janssen cilag grants, personal fees, non-financial support, consultancy from Gilead sciences, grants, personal fees, non-financial support, consultancy from ViiV healthcare, outside the submitted work. Paolo Bonfanti reports personal fees from ViiV, Gilead, Jannsen, and Merck, outside the submitted work. The authors report no other potential conflicts of interest for this work., (© 2020 Squillace et al.)

We report here in silico repurposing studies on 52 new pyridazinone-based small-molecules through inverse virtual screening (iVS) methodologies. These analogues were originally designed as formyl peptide receptor (FPR) ligands. As it is sometimes the case in drug discovery programmes, subsequent biological screening demonstrated the inefficacy of the molecules in binding FPRs, failing in the identification of new hits. Through a focussed drug-repurposing approach we have defined a variety of potential targets that are suitable to interact with this library of pyridazinone-based analogues. A two-step approach has been conducted for computational analysis. Specifically, the molecules were initially processed through a pharmacophore-based screening. Secondly, the resulting features of binding were investigated by docking studies and following molecular dynamic simulations, in order to univocally confirm "pyridazinone-based ligand-target protein" interactions. Our findings propose aspartate aminotransferase as the most favourable repurposed target for this small-molecule series, worth of additional medicinal chemistry investigations in the field.

The insecticidal activity is the result of a series of complex interactions between toxic substances as ligands and insect's enzymes as targets. Actually, synthetic insecticides used in pest control programs are harmful to the environment and may affect non-target organisms; thus, the use of natural products as pest control agents can be very attractive. In the present work, the toxic effect of aniseed ( Pimpinella anisum L.) essential oil (EO) and its nanoemulsion (NE) against the red flour beetle Tribolium castaneum , has been evaluated. To assess the EO mode of action, the impact of sub-lethal concentrations of aniseed EO and NE was evaluated on enzymatic and macromolecular parameters of the beetles, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, total lipids and glucose. Finally, a molecular docking study was conducted to predict the mode of action of the major EO and NE components namely E -anethole, Limonene, alpha-himalachalene, trans-Verbenol and Linalool at binding site of the enzymes AST and ALT. Herein, the binding location of the main compounds in both proteins are discussed suggesting the possible interactions between the considered enzymes and ligands. The obtained results open new horizons to understand the evolution and response of insect-plant compounds interactions and their effect predicted at the molecular levels and side effects of both animal and human.

Macrophage-mediated inflammation is a key pathophysiological component of cardiovascular diseases, but the underlying mechanisms by which the macrophage regulates inflammation have been unclear. In our study, we, for the first time, showed an endogenous sulfur dioxide (SO 2 ) production in RAW267.4 macrophages by using HPLC and SO 2 -specific fluorescent probe assays. Moreover, the endogenous SO 2 generating enzyme aspartate aminotransferase (AAT) was found to be expressed by the macrophages. Furthermore, we showed that AAT2 knockdown triggered spontaneous macrophage-mediated inflammation, as represented by the increased TNF-α and IL-6 levels and the enhanced macrophage chemotaxis; these effects could be reversed by the treatment with a SO 2 donor. Mechanistically, AAT2 knockdown activated the NF-κB signaling pathway in macrophages, while SO 2 successfully rescued NF-κB activation. In contrast, forced AAT2 expression reversed AngII-induced NF-κB activation and subsequent macrophage inflammation. Moreover, treatment with a SO 2 donor also alleviated macrophage infiltration in AngII-treated mouse hearts. Collectively, our data suggest that macrophage-derived SO 2 is an important regulator of macrophage activation and it acts as an endogenous "on-off switch" in the control of macrophage activation. This knowledge might enable a new therapeutic strategy for cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Distinctive from their normal counterparts, cancer cells exhibit unique metabolic dependencies on glutamine to fuel anabolic processes. Specifically, pancreatic ductal adenocarcinoma (PDAC) cells rely on an unconventional metabolic pathway catalyzed by aspartate transaminase 1 (GOT1) to rewire glutamine metabolism and support nicotinamide adenine dinucleotide phosphate (NADPH) production. Thus, the important role of GOT1 in energy metabolism and Reactive Oxygen Species (ROS) balance demonstrates that targeting GOT1 may serve as an important therapeutic target in PDAC., Methods: To assay the binding affinity between Aspulvinone O (AO) and GOT1 proteins, the virtual docking, microscale thermophoresis (MST), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) methods were employed. GOT1 was silenced in several PDAC cell lines. The level of OCR and ECR were assayed by seahorse. To evaluate the in vivo anti-tumor efficacy of AO, the xenograft model was built in CB17/scid mouse., Results: Screening of an in-house natural compound library identified the AO as a novel inhibitor of GOT1 and repressed glutamine metabolism, which sensitizes PDAC cells to oxidative stress and suppresses cell proliferation. Virtual docking analysis suggested that AO could bind to the active site of GOT1 and form obvious hydrophobic interaction with Trp141 together with hydrogen bonds with Thr110 and Ser256. Further in vitro validation, including MST, CETSA and DARTS, further demonstrated the specific combining capacity of AO. We also show that the selective inhibition of GOT1 by AO significantly reduces proliferation of PDAC in vitro and in vivo., Conclusions: Taken together, our findings identify AO as a potent bioactive inhibitor of GOT1 and a novel anti-tumour agent for PDAC therapy.

Mesona blumes polysaccharide (MBP), a primary active component extracted from Mesona blumes, has a number of bioactivities. Nevertheless, hepatoprotective activity of MBP has been rarely reported. The purpose of this study is to investigate hepatoprotective effects of MBP on acute liver injury in mice. Results indicated that the MBP could remarkably decrease the increased levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum caused by tetrachloride (CCl 4 ) treatment (P < 0.05). Medium and high dose of MBP treatment (200 mg/kg body weight, 300 mg/kg body weight) not only prominently enhanced the levels of antioxidant enzymes (superoxide dismutase, SOD) and non-enzyme antioxidants (glutathione, GSH) compared with CCl 4 -induced, but also dramatically decreased lipid peroxidation levels of liver tissues (P < 0.05). In addition, medium and high doses of MBP significantly enhanced the serum levels of IL-1β and TNF-α (P < 0.05). This study showed that MBP had hepatoprotective activity against acute liver injury caused by CCl 4 ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An emerging method to help elucidate the mode of action of experimental drugs is to use untargeted metabolomics of cell-systems. The interpretations of such screens are however complex and more examples with inhibitors of known targets are needed. Here two T-cell lines were treated with an inhibitor of aspartate aminotransferase and analyzed with untargeted GC-MS. The interpretation of the data was enhanced by the use of two different cell-lines and supports aspartate aminotransferase as a target. In addition, the data suggest an unexpected off-target effect on glutamate decarboxylase. The results exemplify the potency of metabolomics to provide insight into both mode of action and off-target effects of drug candidates., Competing Interests: The authors have declared that no competing interests exist.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is extremely refractory to the therapeutic approaches that have been evaluated to date. Recently, it has been demonstrated that PDAC tumors are dependent upon a metabolic pathway involving aspartate aminotransferase 1, also known as glutamate-oxaloacetate transaminase 1 (GOT1), for the maintenance of redox homeostasis and sustained proliferation. As such, small molecule inhibitors targeting this metabolic pathway may provide a novel therapeutic approach for the treatment of this devastating disease. To this end, from a high throughput screen of ∼800,000 molecules, 4-(1H-indol-4-yl)-N-phenylpiperazine-1-carboxamide was identified as an inhibitor of GOT1. Mouse pharmacokinetic studies revealed that potency, rather than inherent metabolic instability, would limit immediate cell- and rodent xenograft-based experiments aimed at validating this potential cancer metabolism-related target. Medicinal chemistry-based optimization resulted in the identification of multiple derivatives with >10-fold improvements in potency, as well as the identification of a tryptamine-based series of GOT1 inhibitors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study examined the effect of chlorpyrifos and/or enrofloxacin on the activity of acetylcholinesterase (AChE) in the blood and brain, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. The experiment was conducted on Wistar strain rats. Chlorpyrifos was administered with a stomach tube at a dose of 0.04 LD50 for 28 days and enrofloxacin at a dose of 5 mg/kg bw for 5 consecutive days. The experiment found that enrofloxacin changed the activity of the enzymes under study only to a small extent. At the dose applied in the experiment, chlorpyrifos decreased the activity of AChE significantly, both in blood and in the brain, and increased the activity of ALT and AST in rat serum. The administration of chlorpyrifos in combination with enrofloxacin changed the activity of the enzymes under study only slightly. A weaker, but longer, inhibition of AChE activity in both blood and the brain was observed in this group compared to the animals exposed only to chlorpyrifos. However, although enrofloxacin, like chlorpyrifos, increases the activity of ALT and AST in serum, their combined administration did not increase the hepatotoxic effect., (Copyright© by the Polish Academy of Sciences.)

Phospholipase A₂s (PLA₂) is a major component of snake venom with diverse pathologic toxicities and, therefore, a potential target for antivenom therapy. Varespladib was initially designed as an inhibitor of mammal PLA₂s, and was recently repurposed to a broad-spectrum inhibitor of PLA₂ in snake venom. To evaluate the protective abilities of varespladib to hemorrhage, myonecrosis, and systemic toxicities that are inflicted by different crude snake venoms, subcutaneous ecchymosis, muscle damage, and biochemical variation in serum enzymes derived from the envenomed mice were determined, respectively. Varespladib treatment showed a significant inhibitory effect to snake venom PLA₂, which was estimated by IC 50 in vitro and ED 50 in vivo. In animal models, the severely hemorrhagic toxicity of D. acutus and A. halys venom was almost fully inhibited after administration of varespladib. Moreover, signs of edema in gastrocnemius muscle were remarkably attenuated by administration of varespladib, with a reduced loss of myonecrosis and desmin. Serum levels of creatine kinase, lactate dehydrogenase isoenzyme 1, aspartate transaminase, and alanine transaminase were down-regulated after treatment with varespladib, which indicated the protection to viscera injury. In conclusion, varespladib may be a potential first-line drug candidate in snakebite envenomation first aid or clinical therapy., Competing Interests: The authors declare no conflict of interest.

Cellular senescence, which leads to a cell cycle arrest of damaged or dysfunctional cells, is an important mechanism to restrain the malignant progression of cancer cells. Because metabolic changes underlie many cell-fate decisions, it has been suggested that cell metabolism might play key roles in senescence pathways. Here, we show that mitochondrial glutamine metabolism regulates senescence in human pancreatic ductal adenocarcinoma (PDAC) cells. Glutamine deprivation or inhibition of mitochondrial aspartate transaminase (GOT2) results in a profound induction of senescence and a suppression of PDAC growth. Glutamine carbon flow through GOT2 is required to create NADPH and to maintain the cellular redox state. We found that elevated reactive oxygen species levels by GOT2 knockdown lead to the cyclin-dependent kinase inhibitor p27-mediated senescence. Importantly, PDAC cells exhibit distinct dependence on this pathway, whereas knockdown of GOT2 did not induce senescence in non-transformed cells. The essentiality of GOT2 in senescence regulation of PDAC, which is dispensable in their normal counterparts, may have profound implications for the development of strategies to treat these refractory cancers.

Both mitochondrial aspartate aminotransferase (mAST) and cytosolic aspartate aminotransferase (cAST) are important components in the malate-aspartate shuttle - one of the two types of NADH shuttles in cells. A major goal of our current study was to determine specifically the roles of cAST in maintaining the [Ca]i, mitochondrial membrane potential and the survival of BV2 microglia by applying molecular approach to modulate the cAST levels. Our study found that decreased cAST by cAST siRNA can lead to significant increases in the [Ca]i, mitochondrial depolarization and apoptosis of BV2 microglia. The cAST siRNA-induced mitochondrial depolarization can be significantly attenuated by an inhibitor of calpain. We further found that the cAST siRNA-induced apoptosis can be prevented by the calpain inhibitor. Collectively, our study suggests that decreased cAST induces calpain activation by increasing the [Ca]i of BV2 microglia, resulting in mitochondrial depolarization and cell death. Moreover, our data suggest that decreased cAST may produce these pathological effects by malate-aspartate shuttle-independent pathways.

Most traditional Chinese medicine prescription dosages are imprecise. This study analyzes the toxicities and adverse effects of a combination the active ingredients of licorice and Kushen medicine: oxymatrine (OMT) and diammonium glycyrrhizinate (DG). The median lethal dose (LD 50 ) and mortality were analyzed in single-dose OMT (or DG) intraperitoneally injected mice with or without combination DG (or OMT). Body weight changes as well as levels of serum sodium and potassium, alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and urea were measured in mice treated with a daily dose of OMT and/or DG for 14days. This study showed that the LD 50 of OMT for males and females were 347.44 and 429.15mg/kg, respectively. The LD 50 of DG were 525.10 and 997.26mg/kg for males and females, respectively. DG significantly decreased the mice LD 50 -induced mortality of the OMT, however OMT did not succeed in reducing the LD 50 -induced mortality rate of DG. The combination of OMT and DG obviously attenuated the changes of the body weight, serum sodium, and potassium induced by DG or OMT alone. These results suggested that toxicity and adverse effects of the OMT was significantly attenuated by DG. The OMT neutralized the adverse effects of the DG, but not the toxicity., (Copyright © 2017. Published by Elsevier Masson SAS.)

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Aims: This study aimed to investigate the inhibitory effects of two natural flavonoids, hesperetin (HT) and hesperidin (HD), on two gluconeogenesis enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and their possible mechanisms of action., Main Methods: Rat liver incubated with different concentrations of HT and HD was used to measure enzyme activities spectrophotometrically, based on monitoring the oxidation of NADH to NAD + at 340nm. Molecular docking simulation was also applied to reveal the molecular mechanism of the inhibition caused by HT and HD., Key Findings: Both flavonoids demonstrated inhibitory effects against the enzyme activities, with IC 50 values of 153.9 and 68.88μM for HT-ALT and HD-ALT treatment respectively. Likewise, the IC 50 values of 85.29μM for HT-AST and 110.3μM for HD-AST were obtained from spectrophotometric results., Conclusion: The docking simulation revealed that HT and HD block the enzyme entrance channel and prevent the substrates from accessing the enzyme active sites. Having prevented production of pyruvate, α-ketoglutarate, and the oxaloacetate, these two compounds inhibit hepatic gluconeogenesis and consequently, hinder the progression of diabetes., Significance: This study suggests that HT and HD may be considered as leading compounds for designing safe and effective drugs in management of increased ALT and AST-related disorders specially diabetes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Previously, we reported the role of synergy between two flavonoids-namely, chrysin and kaempferol-in inhibiting the secretion of a few major proinflammatory mediators such as tumor necrosis factor -alpha (TNF-α), prostaglandin E₂ (PGE₂) , and nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW 264.7 cells. The present study aims to evaluate the effects of this combination on a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Severe sepsis was induced in male ICR mice ( n = 7) via the CLP procedure. The effects of chrysin and kaempferol combination treatment on septic mice were investigated using a 7-day survival study. The levels of key proinflammatory mediators and markers-such as aspartate aminotransferase (AST), TNF-α, and NO-in the sera samples of the septic mice were determined via ELISA and fluorescence determination at different time point intervals post-CLP challenge. Liver tissue samples from septic mice were harvested to measure myeloperoxidase (MPO) levels using a spectrophotometer. Moreover, intraperitoneal fluid (IPF) bacterial clearance and total leukocyte count were also assessed to detect any antibacterial effects exerted by chrysin and kaempferol, individually and in combination. Kaempferol treatment improved the survival rate of CLP-challenged mice by up to 16%. During this treatment, kaempferol expressed antibacterial, antiapoptotic and antioxidant activities through the attenuation of bacterial forming units, AST and NO levels, and increased polymorphonuclear leukocyte (PMN) count in the IPF. On the other hand, the chrysin treatment significantly reduced serum TNF-α levels. However, it failed to significantly improve the survival rate of the CLP-challenged mice. Subsequently, the kaempferol/chrysin combination treatment significantly improved the overall 7-day survival rate by 2-fold-up to 29%. Kaempferol and chrysin revealed some synergistic effects by acting individually upon multiple pathophysiological factors involved during sepsis. Although the kaempferol/chrysin combination did not exhibit significant antibacterial effects, it did exhibit anti-inflammatory and antioxidant activities, which translate to significant improvement in the survival rate of septic animals. These findings suggest the potential application of this combination treatment as a beneficial adjuvant supplement strategy in sepsis control.

The present study was designed to determine whether sulfur dioxide (SO2) could be endogenously produced in adipocyte and served as a novel adipocyte-derived inflammatory inhibitor. SO2 was detected in adipose tissue using high-performance liquid chromatography with fluorescence detection. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) mRNA and protein expressions in adipose tissues were measured. For in vitro study, 3T3-L1 adipocytes were cultured, infected with adenovirus carrying AAT1 gene or lentivirus carrying shRNA to AAT1, and then treated with tumor necrosis factor-α (TNF-α). We found that endogenous SO2/AAT pathway existed in adipose tissues including perivascular, perirenal, epididymal, subcutaneous and brown adipose tissue. AAT1 overexpression significantly increased SO2 production and inhibited TNF-α-induced inflammatory factors, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) secretion from 3T3-L1 adipocytes. By contrast, AAT1 knockdown decreased SO2 production and exacerbated TNF-α-stimulated MCP-1 and IL-8 secretion. Mechanistically, AAT1 overexpression attenuated TNF-α-induced IκBα phosphorylation and degradation, and nuclear factor-κB (NF-κB) p65 phosphorylation, while AAT1 knockdown aggravated TNF-α-activated NF-κB pathway, which was blocked by SO2. NF-κB inhibitors, PDTC or Bay 11-7082, abolished excessive p65 phosphorylation and adipocyte inflammation induced by AAT1 knockdown. This is the first report to suggest that endogenous SO2 is a novel adipocyte-derived inflammatory inhibitor.

Triple-negative breast cancer (TNBC) cell lines are identified to overexpress aspartate transaminase (GOT1), which can potentially control the intracellular levels of reactive oxygen species (ROS) through NADPH synthesis and enhances tumor growth. In this study, the impact of GOT1 on the efficacy of doxorubicin was investigated. Following doxorubicin administration, TNBC cells acquire metabolic alteration, causing increased glutamine flux for the synthesis of aspartate which can be converted into OAA by GOT1. Subsequently, this OAA is converted into malate and then pyruvate, maintaining the NADP(+)/NADPH ratio which neutralize doxorubicin-induced oxidative stress. Repression of GOT1 using the shRNAs for GOT1 resulted in doxorubicin-induced formation of ROS, thereby increasing doxorubicin sensitivity. The enhanced efficacy of doxorubicin by simultaneous repression of GOT1 was also indicated in an in vivo tumor model of TNBC. These results demonstrate that targeting GOT1 in TNBCs may provide a novel therapeutic approach for improving the efficacy of chemotherapy in patients with these refractory tumors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) cells utilize a novel non-canonical pathway of glutamine metabolism that is essential for tumor growth and redox balance. Inhibition of this metabolic pathway in PDAC can potentially synergize with therapies that increase intracellular reactive oxygen species (ROS) such as radiation. Here, we evaluated the dependence of pancreatic cancer stem cells (PCSCs) on this non-canonical glutamine metabolism pathway and researched whether inhibiting this pathway can enhance radiosensitivity of PCSCs. We showed that glutamine deprivation significantly inhibited self-renewal, decreased expression of stemness-related genes, increased intracellular ROS, and induced apoptosis in PCSCs. These effects were countered by oxaloacetate, but not α-ketoglutarate. Knockdown of glutamic-oxaloacetic transaminase dramatically impaired PCSCs properties, while glutamate dehydrogenase knockdown had a limited effect, suggesting a dependence of PCSCs on non-canonical glutamine metabolism. Additionally, glutamine deprivation significantly increased radiation-induced ROS and sensitized PCSCs to fractionated radiation. Moreover, transaminase inhibitors effectively enhanced ROS generation, promoted radiation sensitivity, and attenuated tumor growth in nude mice following radiation exposure. Our findings reveal that inhibiting the non-canonical pathway of glutamine metabolism enhances the PCSC radiosensitivity and may be an effective adjunct in cancer radiotherapy.

This study investigated effects of dietary supplementation with vitamin C, vitamin E on performance, biochemical parameters, and oxidative stress induced by copper toxicity in broilers. A total of 240, 1-day-old, broilers were assigned to eight groups with three replicates of 10 chicks each. The groups were fed on the following diets: control (basal diet), vitamin C (250 mg/kg diet), vitamin E (250 mg/kg diet), vitamin C + vitamin E (250 mg/kg + 250 mg/kg diet), and copper (300 mg/kg diet) alone or in combination with the corresponding vitamins. At the 6th week, the body weights of broilers were decreased in copper, copper + vitamin E, and copper + vitamin C + vitamin E groups compared to control. The feed conversion ratio was poor in copper group. Plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase activities, iron, copper concentrations, and erythrocyte malondialdehyde were increased; plasma vitamin A and C concentrations and erythrocyte superoxide dismutase were decreased in copper group compared to control. Glutathione peroxidase, vitamin C, and iron levels were increased; aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and copper levels were decreased in copper + vitamin C group, while superoxide dismutase, glutathione peroxidase, and vitamin E concentrations were increased; aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were decreased in copper with vitamin E group compared to copper group. The vitamin C concentrations were increased; copper, uric acid, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and malondialdehyde were decreased in copper + vitamin C + vitamin E group compared to copper group. To conclude, copper caused oxidative stress in broilers. The combination of vitamin C and vitamin E addition might alleviate the harmful effects of copper as demonstrated by decreased lipid peroxidation and hepatic enzymes.

Mercury(II) is a highly toxic environmental pollutant leading to oxidative stress in animals and human beings. In this study we aimed to investigate the possible protective effect of a water-soluble polysaccharide (AEP-w1) from the root bark of Aralia elata against experimental mercury(II)-induced cardiovascular oxidative injury in rat model. The results showed that delayed AEP-w1 supplement to HgCl2-treated mice not only decreased serum lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels, but also increased serum nitric oxide (NO) metabolite levels and antioxidant capacity. Moreover, AEP-w1 administration to HgCl2-treated mice significantly decreased malondialdehyde (MDA) level and myeloperoxidase (MPO) activity and increased superoxide dismutase (SOD) and catalase (CAT) activities, along with glutathione (GSH) level in rat cardiac tissue. In addition, elevated serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cr) levels in the saline-treated Hg group were also reversed by AEP-w1 treatment. Therefore, the present study demonstrates that alleviation of HgCl2-induced oxidative injury in rat by AEP-w1 contributes to better understanding of its beneficial effect against cardiovascular diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sulfur dioxide (SO2) is naturally synthesized by glutamate-oxaloacetate transaminase (GOT) from L-cysteine in mammalian cells. We aim to investigate the role of SO2 in inflammation in acute lung injury (ALI) following limb ischemia/reperfusion (I/R). Male Wistar rats were subjected to limb I/R and were injected with saline, GOT inhibitor hydroxamate (HDX, 0.47 mmol/kg), or the SO2 donor Na2 SO3 /NaHSO3 (0.54 mmol/kg/0.18 mmol/kg). Compared with the sham operation, the plasma SO2 levels were significantly decreased by limb I/R treatment. In addition, SO2 concentration and GOT activity in the lung tissue were also reduced in ALI. The occurrence of ALI following limb I/R can be prevented by Na2 SO3 /NaHSO3 treatment, whereas it can be significantly aggravated by HDX. The plasma IL-1β, IL-6, and IL-10 levels were consistent with myeloperoxidase activity and inflammation in lung tissue. In conclusion, our data suggest that downregulation of endogenous SO2 production might be involved in pathogenesis of ALI following limb I/R in rats., (© 2013 Wiley Periodicals, Inc.)

Tartary buckwheat (Fagopyrum tataricum) is a healthy and nutritionally important food item. In this study, we investigated the hepatoprotective effects of 75% ethanol extracts from tartary buckwheat (EEB) against ethanol- and carbon tetrachloride (CCl(4))-induced liver damage. EEB were administered to C57BL/6 mice (ethanol induction) and Sprague-Dawley (SD) rats (CCl(4) induction) for 4 and 8 consecutive weeks, respectively. The major active compounds, rutin and quercetin, were also administered to ethanol- and CCl(4)-induced animals. EEB inhibited increase in serum aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) levels in the ethanol- and CCl(4)-induced animals; similar effects were found after rutin and quercetin administration. Moreover, EEB elevated the antioxidant enzyme activities, including those of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD), and inhibited the levels of hepatic inflammation in the ethanol- and CCl(4)-treated animals. This study suggests that EEB exerts hepatoprotection via promoting anti-oxidative and anti-inflammatory properties against oxidative liver damage.

The organophosphorus pesticide poisoning of the silkworm Bombyx mori is one of the major events causing serious damage to sericulture. Added low-dose rare earths are demonstrated to increase resistance in animals. However, very little is known about whether or not added CeCl₃ can increase resistance of silkworm to phoxim poisoning. The present findings suggested that added CeCl₃ to mulberry leaves markedly increased contents of protein, glucose and pyruvate, and carbohydrate metabolism-related enzyme activities, including lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and attenuated free amino acids, urea, uric acid and lactate levels and inhibited the protein metabolism-related enzymes activities, such as protease, alanine aminotransferase and aspartate aminotransferase in the haemolymph of B. mori, under phoxim toxicity. These findings suggest that added CeCl₃ may improve protein and carbohydrate metabolisms, thus leading to increases of growth and survival rate of B. mori under phoxim stress.

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and l-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding that α-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings' mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

The release of pro-inflammatory cytokines in both acute (IL-1β and TNF-α) and chronic [high mobility group box 1 protein (HMGB1)] phases, is thought to play important roles in the development of fulminant hepatitis (FH). Triterpenoid Acankoreanogenin A (AA) which is extracted from the leaves of the Acanthopanax gracilistylus W.W. Smith (AGS) has shown its inhibiting effect on TNF-α, IL-1β and HMGB1 release in vitro in our preliminary experiments. In present study, we investigated the effect of AA on mice with fulminant hepatitis in vivo. Fulminant hepatitis mice model was established by intraperitoneally injecting galactosamine (GalN) and lipopolysaccharide (LPS). The levels of serum of TNF-α, IL-1β, ALT, AST and HMGB1 from AA-treated mice were measured at different time points. Our results demonstrated that pre-treatment of mice with AA markedly reduced the serum levels of TNF-α, IL-1β, HMGB1, ALT and AST with the improvement in histological features. And the survival rate from AA-treated fulminant hepatitis mice was increased. Furthermore, delayed administration of AA after peak occurrence of the early pro-inflammatory cytokines still endowed significant protection against GalN/LPS-induced lethality. The post-treatment of AA could significantly attenuate the release of HMGB1, but not the TNF-α and IL-1β. These results indicate that AA inhibits the systemic release of pro-inflammatory cytokine HMGB1, and dose-dependently rescue the mice from lethal GalN/LPS-induced fulminant hepatitis, which suggests this component as a candidate therapy for fulminant hepatitis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.

Glutamate is a major neurotransmitter in the CNS but is also a key metabolite intimately coupled to amino acid production/degradation. We consider the effect of inhibition of two key glutamate metabolic enzymes: glutamine synthetase (GS) and aspartate aminotransferase on retinal function assessed using the electroretinogram to consider photoreceptoral (a-wave) and post-receptoral (b-wave) amplitudes. Quantitative immunocytochemistry was used to assess amino acid levels within photoreceptors, ganglion and Müller cells secondary to GS inhibition. Intravitreal injections of methionine sulfoximine reduced GS immunoreactivity in the rat retina. Additionally, glutamate and its precursor aspartate was reduced in photoreceptors and ganglion cells, but elevated in Müller cells. This reduction in neuronal glutamate was consistent with a deficit in neurotransmission (-75% b-wave reduction). Exogenous glutamine supply completely restored the b-wave, whereas other amino acid substrates (lactate, pyruvate, alpha-ketoglutarate, and succinate) only partially restored the b-wave (16-20%). Inhibition of the aminotranferases using aminooxyacetic acid had no effect on retinal function. However, aminooxyacetic acid application after methionine sulfoximine further reduced the b-wave (from -75% to -92%). The above data suggest that de novo glutamate synthesis involving aspartate aminotransferase can partially sustain neurotransmission when glutamate recycling is impaired. We also show that altered glutamate homeostasis results in a greater change in amino acid distribution in ganglion cells compared with photoreceptors.

N-acetylaspartate (NAA) is synthesized predominantly in neurons, and brain homogenate subfractionation studies suggest that biosynthesis occurs at both microsomal (cytoplasmic) and mitochondrial sites. We investigated NAA synthesis in SH-SY5Y human neuroblastoma cells using distinct metabolic precursors that are preferentially metabolized in mitochondria and cytoplasm. Incorporation of (14)C-aspartate and (14)C-malate into NAA was examined in the presence and absence of an inhibitor (aminooxyacetic acid, AOAA) of aspartate aminotransferase (AAT), a central enzyme involved in the biosynthesis of aspartate in mitochondria, and degradation of aspartate in the cytoplasm. AOAA increased the incorporation of (14)C-aspartate into NAA, reflecting direct aspartate-->NAA synthesis in an extramitochondrial location. As expected, AOAA decreased incorporation of (14)C-malate into NAA, reflecting NAA synthesis in mitochondria via the malate-->oxaloacetate-->aspartate-->NAA pathway. When (14)C-malate was used as substrate, the (14)C-NAA/(14)C-aspartate ratio was over 20-fold higher than the ratio obtained with (14)C-aspartate. Because NAA can only be synthesized from aspartate, the higher (14)C-NAA/(14)C-aspartate (product/substrate) ratio obtained with (14)C-malate suggests greater NAA biosynthetic activity. We conclude that NAA biosynthesis occurs in both the cytoplasm and mitochondria of SH-SY5Y cells, and that the contribution from the mitochondrial compartment is quantitatively larger than that in the extramitochondrial compartment.

Previous results showed that pyrazole potentiates lipopolysaccharide (LPS)-induced liver injury in mice. Mechanisms involved the overexpression of cytochrome P450 2E1 (CYP2E1), oxidative stress, and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The current study was carried out to test the hypothesis that the mitochondria permeability transition (MPT) plays a role in this pyrazole plus LPS toxicity. Mice were injected intraperitoneally with pyrazole for 2 days, followed by a challenge with LPS with or without treatment with cyclosporin A (CsA), an inhibitor of the MPT. Serum alanine aminotransferase and aspartate aminotransferase were increased by pyrazole plus LPS treatment, and CsA treatment could attenuate these increases. CsA also prevented pyrazole plus LPS-induced hepatocyte necrosis. Formation of 4-hydroxynonenal protein adducts and 3-nitrotyrosine protein adducts in liver tissue was increased by the pyrazole plus LPS treatment, and CsA treatment blunted these increases. Swelling, cytochrome c release from mitochondria to the cytosol, and lipid peroxidation were increased in mitochondria isolated from the pyrazole plus LPS-treated mice, and CsA treatment prevented these changes. CsA did not prevent the increased levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), pp38 MAPK, and p-JNK2. In conclusion, although CsA does not prevent elevations in upstream mediators of the pyrazole plus LPS toxicity (iNOS, TNF-alpha, CYP2E1, MAPK), it does protect mice from the pyrazole plus LPS-induced liver toxicity by preventing the MPT and release of cytochrome c and decreasing mitochondrial oxidative stress. These results indicate that mitochondria are the critical targets of pyrazole plus LPS in mediating liver injury.

We have investigated the role of lipid per oxidation in pathogenesis of hepatic enzymatic dysfunction during peritoneal intoxication. The investigations were carried out on 36 albino rats. I group were included intact animals, II group were included animals that peritoneal exudates reached with products of lipid per oxidation were injected to abdominal cavity and III group were included reached with animals that superoxiddismutase together with products of lipid per oxidation were injected to abdominal cavity. In all group of animals the concentration of AST, ALT, LDH, alkaline phosphates, GGT enzymes were studied and the results were compared. It was revealed that after injection peritoneal exudates to abdominal cavity, the concentration of AST is decreased and the other (ALT, LDH, alkaline phosphates, GGT) concentration are increased. In further days the blood concentration of all enzymes reached to maximal level. Additionally the injection of superoxiddismutase solution significantly decreases the concentration of these enzymes. It confirms the role of superoxiddismutase in prevention of poliorgan incipiency developing in peritonitis, as well ass in correction of hepatic dysfunction.

Reserpine, a natural product extracted from Rauwolfia serpintina or Rauwolfia vomitoria, is a known dopamine depleter that inhibits several neurotransmitters. Reserpine has been used clinically to control hypertension, schizophrenia, insomnia and insanity. The use of this drug, however, has been limited because of its side effects which include oxidative damage to organs, including the liver. Green tea catechins are potent antioxidants that have the potential to counteract reserpine induced oxidative stress. This study investigated the merits of administering green tea concurrently with reserpine to prevent oxidative hepatic damage in Sprague-Dawely (SD) rats. Reserpine was found to cause hepatic damage, with elevated levels of oxidative stress markers, such as Thiobarbituric Acid Reactive Substances (TBARS), transaminases and cholesterol. Reserpine also induced hepatic ultra-structural damage in the cytoplasmic membrane, nuclear envelope, endoplasmic reticulum (rER), ribosomal stripping and mitochondria. Electron microscopy examination showed revival of liver cells as a result of green tea extract administration to experimental rats.

The effects of a sublethal concentration (7.66 mg/L) of carbofuran, were assessed on Clarias batrachus. The fish were exposed to 7.66 mg of carbofuran/L for 6 days. After 6 days, fish were released into carbofuran-free water in order to study the recovery pattern. Proteins were decreased in gill and kidney and recovery was greater in gill than in kidney. Total amino acids were increased in both tissues. Ammonia level declined in gill and enhanced in kidney throughout the study period. The activities of all enzymes measured were induced in both tissues, except for aspartate aminotransaminase, which was inhibited in gill tissues.

Introduction: Glycolysis is increased in breast adenocarcinoma cells relative to adjacent normal cells in order to produce the ATP and anabolic precursors required for survival, growth and invasion. Glycolysis also serves as a key source of the reduced form of cytoplasmic nicotinamide adenine dinucleotide (NADH) necessary for the shuttling of electrons into mitochondria for electron transport. Lactate dehydrogenase (LDH) regulates glycolytic flux by converting pyruvate to lactate and has been found to be highly expressed in breast tumours. Aspartate aminotransferase (AAT) functions in tandem with malate dehydrogenase to transfer electrons from NADH across the inner mitochondrial membrane. Oxamate is an inhibitor of both LDH and AAT, and we hypothesised that oxamate may disrupt the metabolism and growth of breast adenocarcinoma cells., Methods: We examined the effects of oxamate and the AAT inhibitor amino oxyacetate (AOA) on 13C-glucose utilisation, oxygen consumption, NADH and ATP in MDA-MB-231 cells. We then determined the effects of oxamate and AOA on normal human mammary epithelial cells and MDA-MB-231 breast adenocarcinoma cell proliferation, and on the growth of MDA-MB-231 cells as tumours in athymic BALB/c female mice. We ectopically expressed AAT in MDA-MB-231 cells and examined the consequences on the cytostatic effects of oxamate. Finally, we examined the effect of AAT-specific siRNA transfection on MDA-MB-231 cell proliferation., Results: We found that oxamate did not attenuate cellular lactate production as predicted by its LDH inhibitory activity, but did have an anti-metabolic effect that was similar to AAT inhibition with AOA. Specifically, we found that oxamate and AOA decreased the flux of 13C-glucose-derived carbons into glutamate and uridine, both products of the mitochondrial tricarboxylic acid cycle, as well as oxygen consumption, a measure of electron transport chain activity. Oxamate and AOA also selectively suppressed the proliferation of MDA-MB-231 cells relative to normal human mammary epithelial cells and decreased the growth of MDA-MB-231 breast tumours in athymic mice. Importantly, we found that ectopic expression of AAT in MDA-MB-231 cells conferred resistance to the anti-proliferative effects of oxamate and that siRNA silencing of AAT decreased MDA-MB-231 cell proliferation., Conclusions: We conclude that AAT may be a valid molecular target for the development of anti-neoplastic agents.

As a mechanism-based inactivator of PLP-enzymes, (S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid (SADTA) was cocrystallized with Escherichia coli aspartate aminotransferase (l-AspAT) at a series of pH values ranging from 6 to 8. Five structural models with high resolution (1.4-1.85 A) were obtained for l-AspAT-SADTA complexes at pH 6.0, 6.5, 7.0, 7.5, and 8.0. Electron densities of the models showed that two different adducts had formed in the active sites. One adduct was formed from SADTA covalently linked to pyridoxal 5'-phosphate (PLP) while the other adduct was formed with the inhibitor covalently linked to Lysine246,1 the active site lysine. Moreover, there is a strong indication based on the electron densities that the occurrence of the two adducts is pH dependent. We conclude that SADTA inactivates l-AspAT via two different mechanisms based on the binding direction of the inactivator. Additionally, the structural models also show pH dependence of the protein structure itself, which provided detailed mechanistic implications for l-AspAT.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.

The edible thorny oyster, Spondylus varius (Mizuiri-shoujou), was found to suppress the carbon tetrachloride-induced increase in serum aspartate and alanine aminotransferase activities in mice. Significant suppressive effects on these enzyme activities were found in the fraction eluted with 75% ethanol from polystyrene gel in a dose-dependent manner. These results suggest that S. varius exerts a protective effect against liver injury.

The alpha-ketoglutarate dehydrogenase complex (KGDHC), a control point of the tricarboxylic acid cycle, is partially inactivated in brain in many neurodegenerative diseases. Potent and specific KGDHC inhibitors are needed to probe how the reduced KGDHC activity alters brain function. Previous studies showed that succinyl phosphonate (SP) effectively inhibits muscle and Escherichia coli KGDHC [Biryukov, A. I., Bunik, V. I., Zhukov, Yu. N., Khurs, E. N., and Khomutov, R. M. (1996) FEBS Lett. 382, 167-170]. To identify the phosphonates with the highest affinity toward brain KGDHC and with the greatest effect in living cells, we investigated the ability of SP and several of its ethyl esters to inhibit brain KGDHC, other alpha-keto acid-dependent enzymes, and KGDHC in intact cells. At a concentration of 0.01 mM, SP and its phosphonoethyl (PESP) and carboxyethyl (CESP) esters completely inhibited isolated brain KGDHC even in the presence of a 200-fold higher concentration of its substrate [alpha-ketoglutarate (KG)], while the diethyl (DESP) and triethyl (TESP) esters were ineffective. In cultured human fibroblasts, 0.01 mM SP, PESP, or CESP produced 70% inhibition of KGDHC. DESP and TESP were also inhibitory in the cell system, but only after preincubation, suggesting the release of their charged groups by cellular esterases. Thus, SP and its monoethyl esters target cellular KGDHC directly, while the di- and triethyl esters are activated in intact cells. When tested on other enzymes that bind KG or related alpha-keto acids, SP had minimal effects and its two esters (CESP and TESP) were ineffective even at a concentration (0.1 mM) 1 order of magnitude higher than that which inhibited cellular KGDHC activity. The high specificity in targeting KGDHC, penetration into cells, and minimal transformation by cellular enzymes indicate that SP and its esters should be useful in studying the effects of reduced KGDHC activity on neuronal and brain function.

Background: Curcumin (a component of turmeric) has long been used as a spice and food-coloring agent. In experimental animals, curcumin has shown anti-diabetic, anti-inflammatory, cytotoxic and anti-oxidant properties., Material/methods: The possible hypolipidemic effect of curcumin was investigated in rats fed a high-cholesterol diet (HCD). The lipid profile and activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed in serum, as well as anti-oxidant parameters in liver tissues., Results: Feeding the animals a high cholesterol diet (HCD) for 7 consecutive days (1 ml 100 g(-1)) resulted in marked hypercholesterolemia, increased serum level of low-density lipoprotein cholesterol (LDL-C), but a decreased serum high-density lipoprotein cholesterol (HDL-C). Curcumin admixed with the diet (0.5% w/w) decreased serum total cholesterol (TC) by about 21% and LDL-C by 42.5%, but it increased serum HDL by 50%. The atherogenic indices (LDL-C/HDL-C and TC/HDL-C) were reduced by 52% and 35%, respectively. Curcumin also decreased the enzyme activities of serum AST and ALT, which were increased in HCD animals., Conclusions: Curcumin showed an obvious hypocholesterolemic effect that could be due to an effect on cholesterol absorption, degradation or elimination, but not due to an anti-oxidant mechanism. This could be supported by the finding in our study that neither HCD nor curcumin-admixed HCD had any effects on the liver content of glutathione (GSH) or superoxide dismutase (SOD) activity. Thus one could argue that ingestion of curcumin-containing spices in the diet, especially one rich in fats, could have a lipid-lowering effect.

The activities of phosphatases and transaminases were studied in muscle and liver of the fresh water fish, Oreochromis mossambicus on exposure to different sublethal concentrations (0.25, 0.5, 0.75 and 1 mgl(-1)) of cartap hydrochloride (insecticidal derivative from marine polycheate) for 96 h. There was an overall decrease in phosphatases and transaminases activity in muscle and liver of the fish subjected to cartap hydrochloride.

Background: Reperfusion of the liver after ischemia induces the expression of the heat shock genes and the synthesis of the heat shock proteins (HSP). We studied the effects of the natural antioxidant ergothioneine (EGT) treatment on the expression of HSP70 in ischemic-reperfused (IR) liver., Methods: Adult male Wistar rats were randomly divided into three groups: Sham group given standard laboratory chow and water for 3 weeks followed by sham operation; Control group given standard laboratory chow and water for 3 weeks followed by liver IR injury; EGT group given standard laboratory chow supplementation l-ergothioneine (1.2 mg/kg/d body weight) administered by gavage and water for 3 weeks followed by liver IR injury. Ten rats from each group were killed to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), tissue malondialdehyde (MDA), HSP70 levels, and histologic changes at 30, 60, and 120 min of reperfusion, respectively. Survival was followed for 1 week., Results: IR caused significant increase in serum AST, ALT, LDH, and tissue MDA levels. As compared with the control group, animals treated with EGT experienced a significant decrease in serum AST, ALT, and LDH levels in all reperfusion periods. Tissue MDA levels in animals receiving EGT were significantly reduced as compared with control group at 30 min and 60 min after reperfusion. After ischemia, reperfusion caused a remarkable production of HSP70 in the control group. When the rats were pretreated with EGT, the levels of HSP70 increased significantly in their livers after reperfusion compared with the control group. Liver injury in the EGT-treated animals was lower to that in the control group. The 7-day survival rate was significantly improved (from 50% to 80%) by EGT pretreatment., Conclusion: HSP70 has been shown to induce tolerance against warm IR injury in rat livers. EGT pretreatment protects the liver from IR injury by over-expression of HSP and the subsequent suppression of lipid peroxidation.

The objective of this study was to investigate the effect of ascorbic acid (AA) on the in vitro cytotoxicity of cypermethrin (CM), and on glutathione (GSH) metabolism in rat hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) leakage were measured, as indicators of hepatic damage, at 1, 15 and 30 min of exposure to CM. Glutathione and the activities of glutathione-S-transferase (GST) and gamma glutamyl transpeptidase (gamma-GT) were also measured. CM hepatotoxicity increased in a time and dose-dependent manner. In the presence of 30 microM CM, ALT and AST also increased 49 and 130% (P < 0.05), respectively, indicating metabolic hepatocyte damage. AA (1 mM) was capable to preserve 100% of cell integrity and modulated ALT and AST. Furthermore, CM induced a 27% reduction in the endogenous antioxidant GSH, and increased 203% GST and 283% gamma-GT (P < 0.05), indicating an oxidative insult. The presence of AA showed chemopreventive capacity against CM, recovering 60% of GSH and a 54% decrease in gamma-GT activity. These results suggest that AA in a 1:33 (CM:AA) ratio can modulate up to 90% of the damage caused to the cells by CM. It also demonstrates that AA can act as a primary antioxidant and hepatoprotector in rat hepatocytes.

The effect of Himoliv (HV) was evaluated in carbon tetrachloride or paracetamol induced hepatotoxicity in rats. Liver necrosis was produced by administering single dose of either carbon tetrachloride (CCl4, 1 ml/kg, 50% v/v with olive oil, s.c.) or paracetamol (PC, 1 g/kg, p.o.). The liver damage was evidenced by elevated levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and serum alkaline phosphatase (ALP) and hepatic thiobarbituric acid reacting substances (TBARS) and superoxide dismutase (SOD). HV pretreatment (0.5 and 1.0 ml/kg, p.o.) significantly (P < 0.001) reduced CCl4 or PC-induced elevations of the levels of SGOT, SGPT, ALP and TBARS, while the reduced concentration of SOD due to CCl4 or PC was reversed. Silymarin (25 mg/ kg, p.o.), a known hepatoprotective drug showed similar results.

The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower K(M)s for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered k(cat)s for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k(cat)/K(M)) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK(a)s, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a K(D) for dimer dissociation of 350 nM compared with the wild type K(D) of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on k(cat)/K(M)(Asp). Effects on k(cat)/K(M)(Phe) are more nearly additive and context independent.

Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT- 2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.