Decreased Lactate Dehydrogenase Activity Research Articles

Lactate dehydrogenase activity in Bacteroides fragilis group strains with induced resistance to metronidazole.

The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8mg/L to 96mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1U/mg and 222.0U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity.

The effect of cell-phone radiation on rabbits: Lymphocyte enzyme-activity data

The effect of a GSM 900/1800 mobile phone, which is a widespread source of electromagnetic radiation of the microwave frequency in the environment, on rabbits was studied at power densities of 5–7 μW/cm2. The biological effect was recorded by a sensitive method for the detection of the physiological regulation of enzyme activity inside lymphocytes in blood smears. Succinate dehydrogenase, which is the most powerful energy-supply enzyme in mitochondria, and lactate dehydrogenase, which is an enzyme of glycolysis, were measured. The lactate dehydrogenase to succinate dehydrogenase activity ratio was also calculated as an analog of the Warburg effect, which demonstrates the relationship between glycolysis and respiration. After 60 min of mobile-phone exposure each day for 11 days at a moderate dose, the emitted radiation induced a threefold increase in succinate dehydrogenase activity and a twofold decrease in lactate dehydrogenase activity. As a result, the lactate dehydrogenase/succinate dehydrogenase activity ratio falls from 15 to 5, thus indicating that respiration is predominant over glycolysis. The changes develop already after the first exposure and reach a maximum in 4 days. The predominance of respiration is usually considered as a beneficial state of an organism. However, continuous activation of respiration by mobile phone exposure may cause damage to the normal restorative processes that are supported by glycolysis during periods of rest.

The effect of subchronic inhalations of nitric oxide on metabolic processes in blood of experimental animals

Metabolic processes were investigated in plasma and erythrocytes of Wistar rats exposed to daily 10-min sessions of NO inhalation for 30 days. These included determination of glucose and lactate, catalase activity, and activities of aldehyde dehydrogenase (ALDH), lactate dehydrogenase (LDH), and catalase. NO inhalation in a concentration of 20 ppm, 50 ppm and 100 ppm caused an increase in glucose and lactate. Inhalation of 100 ppm NO also increased catalase activity. Inhalation of all NO concentrations resulted in a decrease of ALDH activity, while the decrease in LDH activity was observed at NO concentrations of 50-100 ppm.

α-Viniferin and resveratrol induced alteration in the activities of some energy metabolism related enzymes in the cestode parasite Raillietina echinobothrida

α-Viniferin (AVF) and its monomer resveratrol (RESV) are natural phytostilbenes produced by several plants in response to injury or under the influence of pathogens such as bacteria or fungi. Our earlier studies have revealed that both the compounds exert anthelmintic activity through alterations of cestode tegument and its associated enzymes. The present study investigates the effects of these phytochemicals on some energy metabolism related enzymes in the fowl tapeworm, Raillietina echinobothrida. The phytostilbenes AVF, RESV and the reference drug praziquantel (PZQ) were tested against some selected enzymes i.e., phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of R. echinobothrida. Exposure of the tapeworm to AVF, RESV and PZQ causes reduction in activity of PEPCK to the extent of 40.57/41.96, 24.58/23.75 and 41.11/13.47%, respectively, and LDH up to 48.95/16.25, 38.31/38.42 and 45.67/41.87%, respectively, at the time of paralysis. Whereas activity of MDH decreased by 34.22/37.7, 39.1/35.24 and 28.83/19.26%, respectively. Decrease in activities of LDH and MDH was also visible through histochemical observations. The results suggest that both the phytochemicals interfere with the energy transducing pathways by inhibiting the studied energy metabolism related enzymes of the parasite.

13C-MR Spectroscopic Imaging with Hyperpolarized [1-13C]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors.

X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized (13)C-MRI enabled detecting metabolic changes in tumors using a tracer [1-(13)C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized (13)C-MRI. SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-(13)C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and (13)C-MR signals were acquired using a 4.7 T scanner. [1-(13)C]pyruvate and [1-(13)C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-(13)C]pyruvate administration. The [1-(13)C]lactate to [1-(13)C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on (13)C-MRI. Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized (13)C-MRI could be useful for the evaluation of early response to radiotherapy. See related commentary by Lai et al., p. 4996.

Intermedin protects against renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress.

BackgroundIntermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. Endoplasmic reticulum stress (ERS) has been implicated in the pathology of renal ischemia/reperfusion (IRI). In the present study, we investigated whether IMD could reduce ERS damage after renal ischemia.MethodsThe kidneys of SD rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. The hypoxia/reoxygenation(H/R) model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. The serum creatinine concentration and lactate dehydrogenase (LDH) activity in the plasma were determined. Histologic examinations of renal tissues were performed with PAS staining. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Additionally, ER staining was used to detect the ERS response.ResultsIn the rat renal IRI model, we found that IMD gene transfer markedly improved renal function and pathology and decreased LDH activity and cell apoptosis compared with the kidneys that were transfected with the control plasmid. IMD significantly attenuated the ERS stress parameters compared with IRI group. Indeed, IMD down-regulated glucose-regulated protein 78 (GRP78), C/EBP homologous protein(CHOP), and caspase 12 protein and mRNA levels. Moreover, in the NRK-52E cell H/R model, IMD overexpression prevented the apoptosis induced by H/R. Furthermore, IMD ameliorated the ER structural changes and concomitantly decreased the levels of GRP78, CHOP and caspase-12.ConclusionThis study revealed that IMD protects against renal IRI by suppressing ERS and ERS-related apoptosis.

Protective effect of gallic acid and Syzygium cumini extract against oxidative stress-induced cellular injury in human lymphocytes

Context: Syzygium cumini (Myrtaceae) presents antioxidant, anti-inflammatory, hypoglycemic and antibacterial effects; however, the cellular and molecular mechanisms of action in the immune system are not yet completely elucidated. Objective: This study evaluates the in vitro effect of gallic acid and aqueous S. cumini leaf extract (ASc) on adenosine deaminase (ADA) and dipeptidyl peptidase IV (DPP-IV) activities, cell viability and oxidative stress parameters in lymphocytes exposed to 2, 2′-azobis-2-amidinopropane dihydrochloride (AAPH). Materials and methods: Lymphocytes were incubated with ASc (100 and 500 µg/ml) and gallic acid (50 and 200 µM) at 37 °C for 30 min followed by incubation with AAPH (1 mM) at 37 °C for 2 h. After the incubation time, the lymphocytes were used for determinations of ADA, DPP-IV and lactate dehydrogenase (LDH) activities, lipid peroxidation, protein thiol (P-SH) group levels and cellular viability by colorimetric methods. Results: (i) HPLC fingerprinting of ASc revealed the presence of catechin, epicatechin, rutin, quercitrin, isoquercitrin, quercetin, kaempferol and chlorogenic, caffeic, gallic and ellagic acids; (ii) for the first time, ASc reduced the AAPH-induced increase in ADA activity, but no effect was observed on DPP-IV activity; (iii) ASc increased P-SH groups and cellular viability and decreased LDH activity, but was not able to reduce the AAPH-induced lipid peroxidation; (iv) gallic acid showed less protective effects than ASc. Discussion and conclusion: ASc affects the purinergic system and may modulate adenosine levels, indicating that the extract of this plant exhibits immunomodulatory properties. ASc also may potentially prevent the cellular injury induced by oxidative stress, highlighting its cytoprotective effects.

Wnt/Glycogen Synthase Kinase 3β/β-catenin Signaling Activation Mediated Sevoflurane Preconditioning-induced Cardioprotection.

Background:Sevoflurane preconditioning (SP) has been shown to invoke potent myocardial protection in animal studies and clinical trials. However, the mechanisms underlying SP are complex and not yet well understood. We investigated the hypothesis that the cardioprotection afforded by SP is mediated via the Wnt/glycogen synthase kinase 3β (GSK3β)/β-catenin signaling pathway.Methods:Two models were established: A Langendorff perfused rat heart model and the H9C2 cell hypoxia/reoxygenation model. Both rats and H9C2 cells were randomly divided into 6 groups as follows: S group, ischemia-reperfusion (I/R) group, DMSO group, IWP group, SP group, and SP + IWP group. Hemodynamic parameters, lactate dehydrogenase (LDH) activity in coronary effluent and cell culture supernatant, and the infarct size were measured to evaluate myocardial ischemia-reperfusion injuries. To determine the activity of Wnt/GSK3β/β-catenin signaling pathway, the expressions of Wnt3a, phospho-GSK3β, and β-catenin were measured by Western blotting.Results:SP improved cardiac function recovery, reduced infarct size (18 ± 2% in the SP group compared with 35 ± 4% in the I/R group; P < 0.05), decreased LDH activity in coronary effluent, and culture supernatant. IWP-2, an inhibitor of Wnt, abolished the cardioprotection by SP. In addition, Western blotting analysis demonstrated that the expressions of Wnt3a, phospho-GSK3β, and β-catenin significantly (P < 0.05) increased in the I/R group, compared with the S group; and compared to I/R group, SP significantly (P < 0.05) increased Wnt3a, phospho-GSK3β, and β-catenin expressions. Pretreatment with IWP-2 significantly (P < 0.05) abolished SP-induced Wnt/GSK3β/β-catenin signaling activation.Conclusions:The results showed for the first time that cardioprotection afforded by SP may be mediated partly via the Wnt/GSK3β/β-catenin signaling pathway.

Inhibition of key markers linked with spermatogenesis and cellular ATP by subchronic exposure to leachate in a rat model.

Few or no studies have measured the toxic effects of subchronic exposure to leachate using key markers linked with spermatogenesis and cellular adenosine triphosphate (ATP) in an experimental rat model. This study was undertaken to evaluate the toxic effects of leachate obtained from the Elewi Odo municipal battery-recycling site (EOMABRL) on male reproductive function using testicular hormones and biomarker of cellular ATP. EOMABRL was administered at 0, 20, 40, 60, 80, and 100% concentrations to adult male rats for 60 days. After exposure, serum was collected for hormonal biochemistry assays, and testes were collected to determine the activity of xanthine oxidase (XO) and lactate dehydrogenase (LDH). Exposure of animals to EOMABRL resulted in a 519.7, 285.7, 569.1, 606.1, and 1,793.2% increase in XO activity with a sequential decrease in LDH activity (marker of cellular ATP) by 44.1, 55.9, 61.4, 69.3, and 89.7%, respectively, compared with the control. Furthermore, EOMABRL caused a significant inhibitory effect on serum luteinizing hormone, follicle-stimulating hormone, and testosterone levels. We conclude that some possible mechanisms by which EOMABRL elicits toxicity in male rat testes could be through inhibition of LDH activity and depletion of serum hormone levels.

Amlodipine prevents adriamycin-induced toxicity in cultured rat mesangial cells by up-regulation of Smad6, Smad7 expression

Extensive studies have demonstrated that transforming growth factor-beta (TGF-β) plays an important role in the progression of renal diseases. A central component of TGF-β is the TGF-β family-specific Smad signal transduction pathway. TGF-β signals through Smad2, 4 to mediate renal fibrosis, whereas induction of Smad6, 7 inhibits renal fibrosis and inflammation. Amlodipine is the most frequently used antihypertensive drug among dihydropyridines. It is beneficial to the kidney and is widely used in treating kidney diseases. The aim of this study was to investigate effects of amlodipine on adriamycin-induced changes of lactate dehydrogenase (LDH) and expression of Smad6, 7 in rat mesangial cells. Results showed that amlodipine (10−8 to 10−5mol/l) significantly decreased LDH activity in rat mesangial cells when given in combination with TGF-β1 (P<0.01); amlodipine (10−7, 10−6mol/l) significantly increased Smad6, 7 mRNA and protein expression in cells treated with adriamycin and TGF-β1 (P<0.01). In conclusion, amlodipine protects against adriamycin-induced toxicity in rat mesangial cells by up-regulation of Smad6, 7 expressions.

Expression Profiles of Myostatin, Myogenin, and Myosin Heavy Chain in Skeletal Muscles of Two Rabbit Breeds Differing in Growth Rate

The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

Reduced Warburg Effect in Cancer Cells Undergoing Autophagy: Steady- State 1H-MRS and Real-Time Hyperpolarized 13C-MRS Studies

Autophagy is a highly regulated, energy dependent cellular process where proteins, organelles and cytoplasm are sequestered in autophagosomes and digested to sustain cellular homeostasis. We hypothesized that during autophagy induced in cancer cells by i) starvation through serum and amino acid deprivation or ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism would be affected, reducing flux to lactate, and that this effect may be reversible. We probed metabolism during autophagy in colorectal HT29 and HCT116 Bax knock-out cells using hyperpolarized 13C-magnetic resonance spectroscopy (MRS) and steady-state 1H-MRS. 24 hr PI103-treatment or starvation caused significant reduction in the apparent forward rate constant (kPL) for pyruvate to lactate exchange compared with controls in HT29 (100 μM PI-103: 82%, p = 0.05) and HCT116 Bax-ko cells (10 μM PI-103: 53%, p = 0.05; 20 μM PI-103: 42%, p<0.0001; starvation: 52%, p<0.001), associated with reduced lactate excretion and intracellular lactate in all cases, and unchanged lactate dehydrogenase (LDH) activity and increased NAD+/NADH ratio following PI103 treatment or decreased LDH activity and unchanged NAD+/NADH ratio following starvation. After 48 hr recovery from PI103 treatment, kPL remained below control levels in HT29 cells (74%, p = 0.02), and increased above treated values, but remained below 24 hr vehicle-treated control levels in HCT116 Bax-ko cells (65%, p = 0.004) both were accompanied by sustained reduction in lactate excretion, recovery of NAD+/NADH ratio and intracellular lactate. Following recovery from starvation, kPL was significantly higher than 24 hr vehicle-treated controls (140%, p = 0.05), associated with increased LDH activity and total cellular NAD(H). Changes in kPL and cellular and excreted lactate provided measureable indicators of the major metabolic processes accompanying starvation- and drug-induced autophagy. The changes are reversible, returning towards and exceeding control values on cellular recovery, which potentially identifies resistance. kPL (hyperpolarized 13C-MRS) and lactate (1H-MRS) provide useful biomarkers for the autophagic process, enabling non-invasive monitoring of the Warburg effect.

Low expression of ERK signaling pathway affecting proliferation, cell cycle arrest and apoptosis of human gastric HGC-27 cells line

This study was carried out for the first time to examine the potential role and the underlying mechanisms of Lycopene in the gastric cancer HGC-27 cells. HGC-27 cells were seeded onto heat-sterilized coverslips in six-well plates and exposed to Lycopene (5, 10, 20, 30 and 40 μmol/L) for periods of 72 h at 37 °C. Results showed that Lycopene (5, 10, 20, 30 and 40 μmol/L) dose-dependently increased NBT positive rate and decreased lactate dehydrogenase activity in HGC-27 cells. In addition, Lycopene (5, 10, 20, 30 and 40 μmol/L) inhibited proliferation and induced G0-G1 phase cell cycle arrest in HGC-27 cells. Western blot and FQRT-PCR analysis showed that Lycopene decreased pERK and extracellular signal-regulated kinase (ERK) protein and mRNA expression in a dose-dependent manner. These findings demonstrate that Lycopene inhibited gastric cancer HGC-27 cells growth and stimulated its apoptosis via the suppressing ERK signaling pathway.

Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.

Impairment of Endothelial-Myocardial Interaction Increases the Susceptibility of Cardiomyocytes to Ischemia/Reperfusion Injury

Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH4) is a required cofactor for nitric oxide (NO) production by endothelial NO synthase (eNOS). Hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH4 content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH4 content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs) were co-cultured with endothelial cells (ECs) and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

Effect of tryptophan on growth, intestinal enzyme activities and TOR gene expression in juvenile Jian carp (Cyprinus carpio var. Jian): Studies in vivo and in vitro

This study was conducted both in vivo and in vitro to investigate the effects of tryptophan on growth performance, digestive and absorptive function and protein synthesis of juvenile Jian carp (Cyprinus carpio var. Jian). 1050 juvenile Jian carp (initial weight 7.73±0.03g) were fed seven isonitrogenous diets with graded concentrations of tryptophan (1.1, 1.7, 2.5, 3.8, 4.9, 6.0, 6.9g/kg diet) for 8weeks. Percent weight gain, feed intake and protein retention value were markedly improved, with increases in dietary tryptophan up to 3.8g/kg diet. Similar trend was found in glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) activities, trypsin, lipase and α-amylase activities, Na+/K+-ATPase, alkaline phosphatase (AKP), γ-glutamyl transpeptidase and creatine kinase activities, and relative expression of eIF4E-binding protein (4E-BP). On the other hand, feed conversion ratio, plasma ammonia concentration and the relative expression of target of rapamycin (TOR) in different tissue showed an opposite pattern. A series of experiments in vitro were then carried out. Compared with the control group, tryptophan supplementation increased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) OD value, protein content and activity of AKP, GOT, GPT and Na+/K+-ATPase in enterocytes and decreased lactate dehydrogenase activity and ammonia concentration in the culture medium. Protein synthesis rate was 17% higher and relative expression of TOR was 28% lower in tryptophan-supplemented than in control carp enterocytes. In conclusion, our results indicate that tryptophan improved fish growth, digestive and absorptive function as well as protein synthesis, which may be partly related to the TOR signaling pathway.

Antitumor effect of blister beetles: An ethno-medicinal practice in Karbi community and its experimental evaluation against a murine malignant tumor model

Ethnopharmacological importanceThe blister beetles Epicauta hirticornis and Mylabris cichorii are used as a folk medicine by the Karbi tribe in Karbi Anglong district of Assam, India for the treatment of different human ailments, including cancer cases. Aim of the studyIt includes field survey related to zoo-therapeutic aspects of two blister beetles in Karbi community, isolation of bio-active compound and evaluation of its antitumor potential with possible mode of action against murine Ehrlich ascites carcinoma (EAC). Materials and methodsThe main bio-active compound of blister beetles was isolated from ethyl acetate extract and the structure was confirmed as cantharidin using NMR, IR, Mass and X-ray diffractometer. The effect of cantharidin on apoptosis, necrosis, autophagy and the apoptosis related signaling pathways were determined using different bioassays, including cell cycle analysis, mitochondrial membrane potential, western blot analysis of cytochrome c, caspases 9, 3/7 assays, and lactate dehydrogenase (LDH) assay. ResultsCantharidin induced apoptosis, necrosis and autophagy cell death in EAC cells. The decrease in mitochondrial membrane potential was observed, which may help to release cytochrome c from mitochondria to cytosol. Cantharidin treatment caused up-regulation of caspases 9 and −3/7 and a decrease in LDH activity in EAC cells. ConclusionThe major bioactive compound of these blister beetles is cantharidin which induces severe apoptosis in EAC cells involving mitochondrial intrinsic pathway. Cantharidin-mediated inhibition of LDH activity may lead to short supply of NAD+ and cut off energy and anabolic supply to cancer cells.

Abstract 419: Two Models of Endothelial Dysfunction: the Importance of Intrinsic Cardioprotective Properties of the Coronary-endothelium

The coronary endothelium plays a major role in protecting cardiomyocytes (CMs) from hypoxia/reoxygenation injury. We are describing two models of endothelial dysfunction compromising the intrinsic cardioprotective properties of endothelial cells (ECs). Tetrahydrobiopterin (BH4) is an essential cofactor for coupling of endothelial nitric oxide synthase (eNOS) to produce the cardioprotective mediator nitric oxide (NO). However, hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to dihydrobiopterin and thereby causing endothelial dysfunction. Additionally, Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. CMs were co-cultured with ECs and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1:3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 μM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate the important intrinsic cardioprotective properties of the coronary endothelium, more specifically increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro

ObjectiveTo determine the cardioprotective effect of magnesium lithospermate B (MLB) on myocardial ischemia/reperfusion (MI/R) injury and to investigate the antioxidant potential in vivo and in vitro. MethodsMI/R injury was induced by the occlusion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and malondialdehyde (MDA). Blood samples were collected to determine serum levels of creatine kinase-MB (CK-MB), cardiac troponin (cTnI) and lactate dehydrogenase (LDH). Furthermore, simulated ischemia/reperfusion (SI/R) injury in vitro was established by oxygen and glucose deprivation (OGD) for 2 h followed by 24-hour recovery period in cardiomyocytes. The activity of LDH in the cultured supernatant and the levels of intracellular reactive oxygen species (ROS), SOD and MDA in cardiomyocytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry. ResultsMLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnI and LDH. Additionally, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomyocytes apoptosis. ConclusionMLB possessed remarkably cardioprotective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.

Oxygen consumption and metabolic responses of freshwater crab Sinopotamon henanense to acute and sub-chronic cadmium exposure

To explore the respiratory and metabolic responses of the freshwater crab (Sinopotamon henanense) to Cd exposure, crabs were acutely exposed to 7.14, 14.28, 28.55mg/L Cd for 96h and subchronically exposed to 0.71, 1.43, 2.86mg/L for three weeks. The oxygen consumption, concentrations of oxyhemocyanin, hemolymph protein, the activities of respiratory enzymes, i.e. lactate dehydrogenase (LDH), NAD-isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO), as well as cco-1(CCO active subunit 1) and ldh mRNA expression level and adenosine triphosphate (ATP) content in crab heart were assessed. Oxygen consumption, concentration of oxyhemocyanin and oxyhemocyanin/blood protein proportion were increased during acute exposure and decreased during sub-chronic exposure. Both exposure schemes induced downregulation of cco-1 gene expression and lowered CCO activity. For acute exposure, tissue ATP level was increased, in association with increased IDH activity and decreased LDH activity, whereas subchronic exposure caused decreased IDH activity accompanied with increased ldh gene expression and LDH activity, resulting in lowered ATP level. By coupling gene expression to biochemical and physiological endpoints, this work provides new insights into the mechanisms involved in metal stress and the differential respiratory and metabolic responses of S. henanense to acute and subchronic Cd exposure.