Isoenzyme Activity Research Articles

N-Acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells

ObjectiveObtaining high number of stem cells is of interest for cell based therapies. N-Acetyl-l-cysteine (NAC) acts as a source of sulfhydryl groups and an anti-oxidative agent. The aim of this study was to test different NAC concentration on proliferation and differentiation of deciduous teeth dental pulp stem cells (DTSCs) in vitro as well as to define the possible underlining mechanism of its effect. DesignNumber of viable, apoptotic and senescent DTSCs was determined after addition of NAC (0.1mM, 1.0mM, 2.0mM). Also, cell cycle analysis, HIF1-α expression, LDH isoenzymes, superoxide-dismutase (SOD) and catalase (CAT) activity, sulfhydryl groups content, the level of lipids’ and proteins’ oxidative damage and differentiation capacity of NAC treated DTSCs was determined. ResultsDTSCs expressed HIF-1α in all conditions. The lowest NAC dose (0.1mM) increased the number of DTSCs by one fifth comparing to the control, most likely stimulating entry of cells into S phase of cell cycle and enhancing the activity of LDH5 isoenzyme. The highest NAC dose (2mM) inhibited DTSCs proliferation. Also, DTSCs had the lowest level of oxidative damage with 0.1mM NAC. All tested NAC concentrations enhanced DTSCs osteo-chondrogenesis. ConclusionThe lowest NAC dose exerted significant positive effect on DTSCs proliferation as well as antioxidative protection creating beneficial environment for stem cells in vitro cultivation especially when their clinical use is important for stimulation of osteo-chondrogenesis.

Pre-treatment of Fulvic Acid Plays a Stimulant Role in Protection of Soybean (Glycine max L.) Leaves Against Heat and Salt Stress

Abstract The purpose of this study was to illuminate the effects of fulvic acid in plants’ stress signaling pathway. 2.0 mg/l fulvic acid was sprayed on soybean leaves for 3 days at 12 h intervals, followed by treatment of 150 mM NaCl or exposed to heat stress at 35°C for 2 h over 2 days. Pre-treatment with fulvic acid increased the relative water content (RWC), antioxidant enzyme, isoenzyme activities (SOD, APX, GST), as well as alleviated the stress-induced oxidative damage by decreasing the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). In addition, the application of fulvic acid under salt stress induced rubisco expression only at 12 h, while it induced the expression of cytochrome c oxidase at 6 h and 12 h. On the other hand, fulvic acid under heat stress induced significant expression of both rubisco and cytochrome c oxidase at 6 h and 12 h. However, under high salinity conditions, fulvic acid suppressed the transcript levels of Hsp70, while it induced increases in Hsp70 levels under heat treatment at 6 h. As a result, in this study, fulvic acid played the role of a regulator and stimulant in stress response of soybean leaves.

Therapeutic drug monitoring of psychotropic medication during pregnancy and lactation

Using psychotropic drugs for the treatment of pregnant and lactating women is a relatively common practice. However, administering psychotropic medication during this period must follow an individual risk-benefit assessment in which the apparent risks associated with fetal or neonatal drug exposure are weighted against the risks of leaving the mental illness untreated. It is not only essential to choose the drugs that bear the lowest risks for the child but also to know the optimal dose. Therapeutic drug monitoring (TDM) is a tool to better individualize dosing regimens. This is of major importance as during pregnancy considerable physiologic changes in absorption, distribution, metabolism and elimination of drugs affecting individual drug concentrations appear. The activity of some CYP isoenzymes and the renal clearance change during pregnancy, both considerably affect the plasma levels of a number of psychotropic drugs. During the breastfeeding period, the factors promoting the transition of psychotropic drugs into the milk need to be considered. However, and more importantly, the metabolism of the individual child determines the infant plasma level and therefore the risk for the child. A TDM routine in patients under psychotropic medication during pregnancy and the breastfeeding period is highly recommended.

Uterine fluid lactate dehydrogenase isoenzyme activity profile

Objective The objective of the present study was to evaluate the diagnostic accuracy of the lactate dehydrogenase (LD) isoenzyme activity profile in uterine fluid, and transvaginal ultrasound (TVS) in the detection of endometrial cancer in women with postmenopausal bleeding. Patients and methods A total of 200 postmenopausal women with one or more episodes of vaginal bleeding were included in the study after signing informed consent. The study was approved by the ethics committee of the Faculty of Medicine, Alexandria University, Egypt. Patients with recent endometrial sampling, current hormone replacement therapy, uterine fluid collection at TVS, or clinical signs of endometritis were excluded from the study. The TVS and uterine fluid sampling for the LD isoenzyme analysis were carried out as office examinations. Endometrial thickness was classified as greater than 5 mm or less than 5 mm on TVS. The LD isoenzyme activity profile was described as normal or abnormal. The LD isoenzyme profile was subsequently related to histopathological diagnosis. Patients with endometrial thickness greater than 5 mm on TVS were evaluated with diagnostic curettage, and those with endometrial thickness less than 5 mm were evaluated with office endometrial biopsy. Results Endometrial carcinoma was found in 18 out of 200 patients (9%). In total, 162 (81%) were found to have endometrium greater than 5-mm thickness on TVS. The LD isoenzyme activity profile was abnormal for 38 (19%) patients. Histopathological diagnosis revealed 18 cases with endometrial cancer and 20 cases with no malignancy. The LD isoenzyme activity profile has 100% sensitivity, 90.1% specificity, 45.4% positive predictive value, 100% negative predictive value, and 88.6% accuracy. TVS for 200 postmenopausal women showed that 162 cases had endometrial thickness greater than 5 mm (81%); 18 of them were diagnosed with endometrial carcinoma (9%) and the remaining 144 cases were diagnosed with benign endometrial pathology (72%); 38 cases had endometrial thickness less than 5 mm (19%) and all of them showed no malignancy (P=0.201). The LD profile showed 38 abnormal results (19%); 18 cases of them diagnosed as endometrial carcinoma (9%) and the remaining 20 cases had benign endometrial pathology (10%). The LD profile showed 162 normal results, all of which had no malignancy (P=0.000*). Conclusion LD isoenzyme profile of uterine fluid could be added as a diagnostic tool for endometrial carcinoma of postmenopausal women. It is a reliable and easy, and is highly tolerable for the patients. At the same time, it can be applied as an office technique. LD isoenzyme profile of uterine fluid could be used as a second step after TVS at a cutoff point of 5 mm, which has proved to be highly reliable. The use of these two methods can reduce the need for curettage and hysteroscopy.

Anticonvulsant Activity of Enantiomeric N-trans-Cinnamoyl Derivatives of 2-Aminopropan-1-ols and 2-Aminobutan-1-ols.

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N-trans-cinnamoyl derivatives of R and S-2-aminopropan-1-ol, as well as R and S-2-aminobutan-1-ol. The structures were confirmed by spectroscopy and for derivatives of 2-aminopropan-1-ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine-induced status prevention. Additionally, derivatives of 2-aminopropan-1-ols were tested in benzodiazepine-resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(-)-(2E)-N-(1-hydroxypropan-2-yl)-3-phenylprop-2-enamide pharmacological parameters were found as follows: ED50 = 76.7 (68.2-81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED50 = 127.2 (102.1-157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD50 = 208.3 (151.4-230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2-aminopropan-1-ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482-488, 2016. © 2016 Wiley Periodicals, Inc.

Upregulated ATF6 contributes to chronic intermittent hypoxia-afforded protection against myocardial ischemia/reperfusion injury.

In the present study, we investigated the role of activating transcription factor 6 (ATF6) in the mechanism by which chronic intermittent hypoxia (CIH) increases tolerance to myocardial ischemia/reperfusion (I/R). Experiments were conducted using a rat model of I/R injury in vivo and isolated Langendorff-perfused rat hearts ex vivo. The role of Akt in this process was also investigated in vitro using rat myoblast H9c2 cells. Cell viability was measured using a cell counting kit-8 assay. Lactate dehydrogenase (LDH) and creatine kinase cardiac isoenzyme activity were also measured as markers of cellular damage. ATF6, Akt and phosphorylated (p)-Akt expression was analyzed by western blot analysis. RNA interference (RNAi) was used to suppress ATF6 expression. We noted that ATF6 expression in the ventricular myocardium was significantly increased in rats exposed to CIH. Furthermore, we noted that CIH preserved cardiac function after I/R in vivo and improved post-ischemic recovery of myocardial performance in isolated rat hearts. ATF6 and p-Akt expression was upregulated in cultured H9c2 cells exposed to chronic mild hypoxia compared with those cultured under normoxic conditions. Chronic mild hypoxia attenuated subsequent simulated I/R injury in H9c2 cells (48 h), as evidenced by increased cell viability and decreased LDH activity. By contrast, decreased cell viability and increased LDH activity were observed in siRNA-ATF6-transfected H9c2 cells, with a concomitant reduction in p-Akt levels. These results indicated that ATF6 upregulation is involved in the mechanism by which CIH attenuates myocardial I/R injury, possibly through upregulation of p-Akt, which is a key regulator of cardiomyocyte survival.

Imidazoacridinone antitumor agent C-1311 as a selective mechanism-based inactivator of human cytochrome P450 1A2 and 3A4 isoenzymes.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311), a promising antitumor agent that is also active against autoimmune diseases, was determined to be a selective inhibitor of the cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1311 might modulate the effectiveness of other drugs used in multidrug therapy. The present work aimed to identify the mechanism of the observed C-1311-mediated inactivation of CYP1A2 and CYP3A4. The inactivation experiments were performed in vitro using the human recombinant CYP1A2 and CYP3A4 (Bactosomes). CYP isoenzyme activities were determined using the CYP-specific reactions, 7-ethoxycoumarin O-deethylation (CYP1A2) and testosterone 6β-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by RP-HPLC with UV-Vis detection. The inhibition of CYPs by C-1311 was time-, concentration- and NADPH-dependent, which suggested a mechanism-based mode of action. Using a 10-fold dilution method and potassium ferricyanide we demonstrated the irreversible nature of the inhibition. In addition, the inhibition was attenuated by the presence of alternate substrates (alternative active site ligands) but not by a nucleophilic trapping agent (glutathione) or a reactive oxygen scavenger (catalase), which further supported a mechanism-based action. Substrate depletion partition ratios of 299 and 985 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. Our results indicated that C-1311 is a potent mechanism-based inactivator of CYP1A2 and CYP3A4. This finding provided new insights into the mechanism of C-1311 antitumor action, particularly in relation to potential pharmacokinetic drug-drug interactions between C-1311 and/or its derivatives and the substrates of CYP isoforms.

Association of G22A and A4223C ADA1 gene polymorphisms and ADA activity with PCOS

ABSTRACTAdenosine deaminase-1 (ADA1) regulates the concentration of adenosine as the main modulator of oocyte maturation. There is compelling evidence for the association of ADA1 gene polymorphisms with many diseases but the importance of ADA1 polymorphisms in polycystic ovary syndrome (PCOS) has not been studied before. This study investigates serum total ADA activity (tADA), ADA1 and ADA2 isoenzyme activities, and genotype and allele frequencies of G22A and A4223C polymorphisms in healthy and PCOS women. In this case-control study 200 PCOS patients and 200 healthy women were enrolled. Genomic DNA was extracted from whole blood and the PCR-RFLP technique was used to determine the G22A and A4223C variants. The genotype frequencies were calculated and the association between polymorphic genotypes and enzyme activities were determined. tADA activity was significantly lower in the PCOS group compared with the control group (27.76±6.0 vs. 39.63±7.48, respectively). PCOS patients also showed reduced activity of ADA1 and ADA2. PCOS was not associated with G22A polymorphism whereas AA, AC, and CC genotypes of A4223C polymorphism were found distributed differently between the control and the PCOS women where the C allele showed a strong protective role for PCOS (odds ratio=1.876, p=0.033). The present study for the first time showed that lower ADA activity may be involved in pathogenesis of PCOS by maintaining a higher concentration of adenosine affecting follicular growth. As a novel finding, we also showed great differences in genotype distribution and allele frequencies of A4223C polymorphism between groups indicating a protective role for C allele against PCOS.AbbreviationsADA: adenosine deaminase PCOS: polycystic ovary syndrome PCR-RFLP: polymerase chain reaction–restriction fragment length polymorphism tADA: total adenosine deaminase

Production, purification, and characterization of a novel cold-active superoxide dismutase from the Antarctic strain Aspergillus glaucus 363

The Antarctic fungal strain Aspergillus glaucus 363 produces cold-active (CA) Cu/Zn-superoxide dismutase (SOD). The strain contains at least one gene encoding Cu/Zn-SOD that exhibited high homology with the corresponding gene of other Aspergillus species. To our knowledge, this is the first nucleotide sequence of a CA Cu/Zn-SOD gene in fungi. An effective laboratory technology for A. glaucus SOD production in 3 L bioreactors was developed on the basis of transient cold-shock treatment. The temperature downshift to 10 °C caused 1.4-fold increase of specific SOD activity compared to unstressed culture. Maximum enzyme productivity was 64 × 103 U kg−1 h−1. Two SOD isoenzymes (Cu/Zn-SODI and Cu/Zn-SODII) were purified to electrophoretic homogeneity. The specific activity of the major isoenzyme, Cu/Zn-SODII, after Q-Sepharose chromatography was 4000 U mg−1. The molecular mass of SODI (38 159 Da) and of SODII (15 835 Da) was determined by electrospray quadropole time-of-flight (ESI-Q-TOF) mass spectrometry and dynamic light scattering (DLS). The presence of Cu and Zn were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The N-terminal amino acid sequence of Cu/Zn-SODII revealed a high degree of structural homology with Cu/Zn-SOD from other fungi, including Aspergillus species.

Ligand-dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family.

Expression of the human CCR2 receptors, CCR2a and CCR2b, in mammalian cells results in ligand-dependent changes in the activity of multiple cellular signal transduction pathways, mediated in most cases by pertussis toxin-sensitive heterotrimeric G proteins of the Gi/o subfamily. In addition, CCR2a and CCR2b receptors have been shown to couple to Gq family members, triggering the canonical activation of phospholipase Cβ isoenzymes. Activation of pertussis toxin-insensitive Gq proteins by cell-surface receptors is not only coupled to activation of phospholipase isoenzymes but also to Rho guanine nucleotide exchange factors, which in turn mediate activation of the Rho GTPases. Activated Rho GTPases regulate numerous cellular functions, including the organization of the actin cytoskeleton and gene transcription, such as the transcription factor serum response factor. These findings prompted us to investigate whether CCR2a and/or CCR2b stimulate serum response factor activity. The results presented herein demonstrate that stimulation of human CCR2a- or CCR2b-expressing COS-7 cells caused a vigorous induction of serum response factor activity. This effect was specifically mediated by Gq and/or G14, as well as Rho A and/or a closely related Rho GTPase. Furthermore, the stimulatory effect of CCR2a and CCR2b and Gαq was sensitive to coexpression of the Gαq-interacting leukemia-associated Rho guanine nucleotide exchange factor. The findings of the work indicate a role for Gαq and/or Gα14 and in CCR2a/CCR2b-stimulated Rho A GTPase-mediated serum response factor activation and introduce a noncanonical pathway activated by CCR2 receptors by coupling to Gq proteins.

Synthesis of novel sulfonamides under mild conditions with effective inhibitory activity against the carbonic anhydrase isoforms I and II

Novel sulfonamide derivatives 6a–i, as new carbonic anhydrase inhibitors which candidate for glaucoma treatment, were synthesized from the reactions of 4-amino-N-(4-sulfamoylphenyl) benzamide 4 and sulfonyl chloride derivatives 5a–i with high yield (71–90%). The structures of these compounds were confirmed by using spectral analysis (FT-IR, 1H NMR, 13C NMR, LC/MS and HRMS). The inhibition effects of 6a–i on the hydratase and esterase activities of human carbonic anhydrase isoenzymes, hCA I and II, which were purified from human erythrocytes with Sepharose®4B-l-tyrosine-p-aminobenzene sulfonamide affinity chromatography, were studied as in vitro, and IC50 and Ki values were determined. The results show that newly synthesized compounds have quite powerful inhibitory properties.

Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic anhydrase isoforms hCA I and hCA II

Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of the 12 synthesized sulfonamide (5a–l) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. Among them 5b was found to be the most active (IC50 = 0.35 μM; Ki: 0.33 μM) for hCA I and hCA II.

Modern aspects of the safe use of extension QT interval medicines

Prolongation of QTinterval is apredictor offatal cardiac arrhythmias and sudden death. In clinical practice, medicines with possible and conditional risk of QTprolongation are combine. The danger of such interactions could be enhanced if medi- cines interact themselves at metabolic rate. The interaction of drugs with possible and conditional risk of QTprolongation and interaction of these drugs with drugs that can influence the metabolic activity of cytochrome P450 isoenzymes require specific attention from physicians. Predictability of QTprolongation by drug-drug interactions at metabolic rate in drug administration will increase the safety of pharmacotherapy of drugs with possible and conditional risk of QTprolongation.

Protective Effects of Polyphenol Extracts from Sea Buckthorn (<i>Hippopha&#235 rhamnoides</i> L.) on Rat Hearts

The objective of present study was to investigate the antioxidative and cardioprotective effects of polyphenol extracts from sea buckthorn (PESB) against myocardial ischemia reperfusion injury (MIRI). PESB was analyzed using HPLC-DAD and their antioxidant activities were evaluated by measuring the intrinsic free radicals cavenging capacity and inhibiting the formation of hydroxyl radical capacity. Experiments on the isolated rat hearts were carried out according to the improved Langendorff retrograde perfusion technology. Coronary effluent of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) activities were analyzed to determine the extent of myocardial injury. Protein expression levels of Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) I and II were measured to evaluate the degree of autophagy. In conclusion, coupled results from both in vivo and in vitro experiments have confirmed that PESB has potential health implications in both prevention and amelioration of MIRI, which may be attributed to the inhibition of autophagy.

Tempol, a Membrane-Permeable Radical Scavenger, Exhibits Anti-Inflammatory and Cardioprotective Effects in the Cerulein-Induced Pancreatitis Rat Model.

To date, it remains unclear whether mild form of acute pancreatitis (AP) may cause myocardial damage which may be asymptomatic for a long time. Pathogenesis of AP-related cardiac injury may be attributed in part to ROS/RNS overproduction. The aim of the present study was to evaluate the oxidative stress changes in both the pancreas and the heart and to estimate the protective effects of 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (tempol) at the early phase of AP. Cerulein-induced AP led to the development of acute edematous pancreatitis with a significant decrease in the level of sulfhydryl (–SH) groups (oxidation marker) both in heart and in pancreatic tissues as well as a substantial increase in plasma creatine kinase isoenzyme (CK-MB) activity (marker of the heart muscle lesion) which confirmed the role of oxidative stress in the pathogenesis of cardiac damage. The tempol treatment significantly reduced the intensity of inflammation and oxidative damage and decreased the morphological evidence of pancreas injury at early AP stages. Moreover, it markedly attenuated AP-induced cardiac damage revealed by normalization of the –SH group levels and CK-MB activity. On the basis of these studies, it is possible to conclude that tempol has a profound protective effect against cardiac and pancreatic damage induced by AP.

CYP3A4 activity and haloperidol effects in alcohol addicts.

Haloperidol is one of the most commonly used typical antipsychotics [2]. It has a powerful antipsychotic activity blocking mesolimbic postsynaptic dopamine receptors. Unwanted adverse effects accompany the use of haloperidol. Therefore alcohol abusers' attitudes towards haloperidol are ambiguous and often negative, which sometimes limits it's use in patients with addictive disorders [3]. Cytosolic carbonyl reductase reduces haloperidol to reduced form, which has 10-20% of the activity of the parent molecule. It is further metabolized by CYP3A4 to a tetrahydropyridine and then conjugated by glucuronidation and sulphation. Reduced haloperidol is back-oxidized to haloperidol by CYP3A4 and CYP2D6. Haloperidol is N-dealkylated by CYP3A4 and CYP2D6 to 4-chlorophenyl-4-hydroxypiperidine and p-fluorobenzoyl propionic acid. The correlation between CYP2D6 and CYP3A4 activity and the rate of biotransformation of haloperidol was demonstrated in a number of studies on patients with schizophrenia [1, 2, 5]. At the same time other studies deny or disaffirm this correlation [4]. To estimate the correlation between CYP3A4 isoenzyme activity and the efficacy and safety of haloperidol in patients with alcohol abuse during the exacerbation of the addiction. The study involved 15 men, alcohol abusers, in exacerbation of their addiction, who were hospitalized in Moscow Research and Practical Centre for Narcology of the Departament of Public Health. All 15 patients received haloperidol in tablets and injections. Determination of CYP3A4 activity was performed using high performance liquid chromatography with mass spectrometry (HPLC/ MS) by determination of endogenous substrate of this isoenzyme and its metabolite in urine - the ratio: cortisol/6-beta-hydroxycortisol. We used international psychometric scales to assess efficacy of haloperidol (the scale of determining the severity of addiction of The National Research Center on Addictions of the Ministry of Health Of Russia, Hamilton Anxiety Research Scale (HARS)). The safety of haloperidol was estimated by the UKU Side-Effect Rating Scale. Scales express the clinical picture of the abuse. The higher the score, the more pronounced the addiction is. Calculating the differences in scores of the scales allowed for clinical assessment of haloperidol effects. The larger the difference in scores was, the more pronounced were the changes in clinical picture of abuse, and the higher the efficacy of therapy was assumed. Statistical analysis of the results of the study was performed by non-parametric statistics by the program STATISTICA v10.0 («StatSoft Inc.», USA). The normality of sample distribution was estimated by Shapiro-Wilk's W-test, and the homogeneity of dispersion, that was estimated by Fisher's T-test (in case of comparison of two samples). The differences were evaluated as significant in case of p < 0,05 (statistic power >80%). To determine the correlation between the quantitative characteristics Spearman rank R coefficient was calculated. The value of correlation coefficient r from 0,3 to 0,7 (p < 0,05) indicated positive moderate, but significant correlation between the characteristics, r>0,7 (p < 0,05) - strong and significant correlation, negative value of r indicated inverse correlation. Data analysis demonstrated a correlation between the activity of isoenzyme CYP3A4 and the scores of pathological addiction (r1 = -0,36), HARS (r2 = -0,45), UKU Side-Effect Rating Scale (r3 = -0.15) in the entire group (p < 0.05). In a group of patients, who received the higher doses of haloperidol (more than 7.5 mg per day in tablets or 5 mg per day in injections), the following results were received in the same groups of data: r1 = -0.68, r2 = -0.71, r3 = -0.76 (p < 0.05). The results demonstrate the correlation between CYP3A4 activities and the efficacy and safety of haloperidol in alcohol abusers during the exacerbation of the addiction. The inverse correlation indicates that the higher the activity of CYP3A4 is, the lower the efficacy of haloperidol is. Also it can be assumed that the presence of strong correlation between the activity of CYP3A4 and the efficacy of haloperidol in group of patients, who received higher doses of haloperidol, may indicate that CYP3A4 is involved in haloperidol metabolism when it is used at higher doses. It should be noted that in this research the activity of CYP3A4 was determined using high performance liquid chromatography with mass spectrometry (HPLC/MS) by determination the ratio of cortisol/6-beta-hydroxycortisol for the fist time. To increase the level of our confidence in the results further studies with a larger number of people are necessary.

Abstract 15022: Alkaline Phosphatase Activity After Infant Cardiac Surgery: Kinetics and Association With Post-operative Cardiovascular Support

Introduction: Alkaline phosphatase (AP) may be protective after CV surgery through dephosphorylation of endotoxin and extracellular adenine nucleotides. AP activity falls after infant CV surgery and low activity is associated with increased support requirements. However, the kinetics of AP loss and recovery, the isoenzymes involved, and the temporal relation of low AP activity to increased support are unknown. We hypothesized loss of all isoenzymes during surgery leads to decreased AP activity and that early low AP activity is associated with increased cardiovascular support at 6-48hr. Methods: Prospective study of 93 infants &lt;120 d/o undergoing CV surgery with bypass. Total/isoenzyme specific AP activity and vasoactive inotropic score (VIS) were obtained pre-operation, pre-separation and at 6, 24, 48 and 72hr after ICU admission. AP activities were determined by commercial assay and concentration by multiplex immunoassay. Low AP activity was defined as ≤80 U/L. Results: Median AP activity decreased through 6hr due to decreased bone and liver 2 isoenzyme activities (fig 1). Liver 1 isoenzyme increased post-bypass then decreased through 72hr. Activity was highly correlated with concentration at all time points (r=0.83-0.89; p&lt;0.0001). Nadir AP activity coincided with peak VIS. Infants with low AP activity at 6hr had significantly higher VIS through 48hr (6hr 12.1 vs 8.1, p&lt;0.0005; 24hr 11.2 vs 5.3, p&lt;0.0001; 48hr 6.9 vs 3.0, p&lt;0.0005). Similar findings occurred with low pre-separation and 24hr AP activity. On multivariate analysis, low AP activity at 6hr was independently associated with higher VIS at 6, 24 and 48hr. Conclusions: Loss of bone and liver 2 isoenzymes leads to decreased AP activity after infant CV surgery. Low early AP activity is associated with increased cardiovascular support requirements 6-48hr later. AP activity may serve as an early biomarker of post-operative hemodynamic instability and a potential therapeutic target in this population.

Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing.

Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

Characterization of Cu/Zn-SOD enzyme activities and gene expression in soybean under low nitrogen stress.

Superoxide dismutase (SOD) plays an important role in antioxidant defense in nearly all cells, and is speculated to be closely related to plant resistance to biotic and abiotic stresses, such as drought, salt, heavy metal and pathogen attack. However, little is known about the effects of SOD activity and its isoenzymes on low nitrogen stress tolerance and its effects on adaptability of plants to nitrogen limitation. Ten SOD isoenzymes were identified in soybean root, stem, leaf and mature seed, and were classified into three families (α.1, β.1-4 and γ.1-5). SOD activity was significantly elevated in soybean leaf and root. Conversely, under low-nitrogen conditions, only β.2 isoenzyme activity, belonging to the Cu/Zn-SOD family, was induced obviously in the root of soybean cultivar cv. WS01-15. Moreover, the expression of three Cu/Zn-SOD genes was analyzed under low nitrogen stress. GmCZ-SOD1 gene was induced significantly in soybean root under low nitrogen stress. Interestingly, evolutionary analysis showed that this gene underwent a strong artificial selection during soybean domestication, suggesting that the Cu/Zn-SOD gene plays an essential role in the adaptive evolution of soybean nitrogen limitation resistance. GmCZ-SOD is important for adaptability of soybean to nitrogen limitation and these results provide useful information to unravel its biological role in low nitrogen resistance in plants. © 2015 Society of Chemical Industry.

Methods for the assessment of isoenzymes activity in sunflower seeds

Enzyme activity detection in seeds allows the assessment of the deterioration process in the early stages. In this context, the objective was to assess the effectiveness and the degree of similarity between the results of izoenzymes activity using different methods, as follows: spectrophotometry, visual analysis after electrophoresis, evaluation of the bands nodes gels with software GelAnalyzer. 250 hybrid seeds were submitted to artificial aging to form four lots with differences in physiological quality. In addition to the enzymatic determinations, physiological germination tests, emergence and emergence speed index were performed. It was concluded that there are differences in results according to the methods used; the results were consistent between test methods and correlated with physiological results when the catalase enzyme was evaluated; and in the isozyme analysis of superoxide dismutase and alcohol dehydrogenase there was a divergence among the results of the methodologies, and according to such, the relationship to seeds physiological quality.