Isoenzyme Activity Research Articles

Pharmacogenetic and Clinical Predictors of Clopidogrel Insufficiency in a Patient with Atherosclerosis Obliterans of the Lower Extremities: Clinical Case

The considered clinical case of combination of multifocal atherosclerotic vascular lesion with type 2 diabetes mellitus and liver fibrosis demonstrates a combination of polyvalent risk factors for resistance to antiplatelet therapy with clopidogrel. The decrease in the effectiveness of prolonged therapy with P2Y 12 inhibitor was clinically manifested by repeated thrombotic events and was confirmed by laboratorial VerifyNow P2Y 12 Assay test system as a low percentage of inhibition of ADP-induced platelet aggregation. We established probable genetic predictors of the decrease in the effectiveness of antiplatelet therapy in this patient, namely, the carriage of polymorphic markers of the ABCB1 CT, CES1 CA and CYP3A4*22 CT genes that determine the decrease in absorption, excessive hydrolysis of the drug and reduced activity of isoenzymes and transporters, that leads to disorders of active clopidogrel metabolite formation. A potential contribution of hepatic dysfunction to reduction in antiaggregant effect of P2Y 12 inhibitor was demonstrated. Variant of drug interaction of clopidogrel and an inhibitor/substrate of P450 CYP2C19 – omeprazole, accompanied by a decrease in the effectiveness of antiplatelet therapy, was also considered.

Adenosine Metabolism in COPD: A Study on Adenosine Levels, 5′-Nucleotidase, Adenosine Deaminase and Its Isoenzymes Activity in Serum, Lymphocytes and Erythrocytes

Adenosine is a signaling molecule which is produced in high concentrations during airway inflammation. Airway inflammation is a characteristic feature of COPD. Therefore, the current study was designed to evaluate the changes in adenosine metabolism in COPD and correlate these changes with severity of the disease.The study was conducted on 50 healthy controls (25 healthy non-smokers and 25 healthy smokers) and 46 COPD patients (21 moderate, 15 severe and 10 very severe). The patients were sub-divided into moderate, severe and very severe categories as per the GOLD spirometric classification. Blood was collected from each subject and serum, lymphocytes and erythrocytes were separated. The adenosine levels and activities of 5′-nucleotidase, adenosine deaminase and its isoenzymes were assessed in serum, lymphocytes and erythrocytes. The data were analyzed statistically. A p value < 0.05 was considered as significant.In healthy smokers and COPD patients the adenosine levels increased. In COPD patients 5′-nucleotidase activity increased significantly in serum, lymphocytes and erythrocytes. The activities of ADA and isoenzymes decreased significantly in serum of healthy smokers and COPD patients, in lymphocytes and erythrocytes of very severe COPD patients and of ADA and ADA2 in lymphocytes and erythrocytes of moderate and severe COPD patients. The FEV1 (% of predicted) showed a significant negative correlation with adenosine levels and 5′-nucleotidase activity in serum, lymphocytes and erythrocytes and significant positive correlation with ADA and isoenzymes activity in serum and lymphocytes of COPD patients.We conclude that the adenosine metabolism changes in COPD. The adenosine levels and 5′-nucleotidase activity increase, and ADA activity decreases with severity of the disease.

LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 and P-glycoprotein probe substrates and their metabolites using subtherapeutic doses

Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC–MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01–5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/*3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D6*4/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.

PO-194 Urinary metabolomics study on effects of Rhodiola on Marathon Amateurs after Quantitative Exercise Load

Objective To study the effect of Chinese medicine Rhodiola on oxidative stress injury in amateur marathon runners after quantitative exercise load (20 km) and explore its mechanism.&#x0D; Methods Eight marathon amateurs were divided into four groups according to different test time, including before and after quantitative exercise load (group C and group CE), before and after quantitative exercise load after taking a month of the Rhodiola (group MC and group ME). The participants had serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), as well as myocardial enzyme index - creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB) and glutamic oxaline aminotransferase (AST/GOT) activity evaluated. In order to further explore the mechanism of action of Rhodiola, the urine was analyzed by 1H-HMR metabonomics technique.&#x0D; Results 1) Compared with group C, the activity of serum CK, LDH and AST/GOT of group CE increased significantly (P&lt;0.01 or P&lt;0.05), MDA content and SOD activity also increased significantly (P&lt;0.05). A total of fifteen potential biomarkers were found in group CE, such as valine, lactic acid, 2- hydroxy isobutyric acid and so on (P&lt;0.05 and VIP&gt;1). 2) Compared with group CE, the activity of serum CK and AST/GOT and the content of MDA of group ME decreased significantly (P&lt;0.05), eleven metabolites among the fifteen potential biomarkers reverted significantly (P&lt;0.01 or P&lt;0.05), which mainly involved in 4 metabolic pathways including alanine, aspartic acid and glutamic acid metabolism and so on.&#x0D; Conclusions Rhodiola can enhance the antioxidant capacity and improve myocardial damage of marathon amateurs after quantitative exercise load, which may be due to increased synthesis and utilization of aminoacyl-tRNA and other amino acids.

Severe steatosis induces portal hypertension by systemic arterial hyporeactivity and hepatic vasoconstrictor hyperreactivity in rats

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease. The presence of portal hypertension has been demonstrated in NAFLD prior to development of inflammation or fibrosis, and is a result of extrahepatic and intrahepatic factors, principally driven by vascular dysfunction. An increased intrahepatic vascular resistance potentially contributes to progression of NAFLD via intralobular hypoxia. However, the exact mechanisms underlying vascular dysfunction in NAFLD remain unknown. This study investigates systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis. Wistar rats were fed a methionine-choline-deficient diet, inducing steatosis, or control diet for 4 weeks. In vivo hemodynamic measurements, aortic contractility studies, and in situ liver perfusion experiments were performed. The mean arterial blood pressure was lower and portal blood pressure was higher in steatosis compared to controls. The maximal contraction force in aortic rings from steatotic rats was markedly reduced compared to controls. While blockade of nitric oxide (NO) production did not reveal any differences, cyclooxygenase (COX) blockade reduced aortic reactivity in both controls and steatosis, whereas effects were more pronounced in controls. Effects could be attributed to COX-2 iso-enzyme activity. In in situ liver perfusion experiments, exogenous NO donation or endogenous NO stimulation reduced the transhepatic pressure gradient (THPG), whereas NO synthase blockade increased the THPG only in steatosis, but not in controls. Alpha-1-adrenergic stimulation and endothelin-1 induced a significantly more pronounced increase in THPG in steatosis compared to controls. Our results demonstrate that severe steatosis, without inflammation or fibrosis, induces portal hypertension and signs of a hyperdynamic circulation, accompanied by extrahepatic arterial hyporeactivity and intrahepatic vascular hyperreactivity. The arterial hyporeactivity seems to be NO-independent, but appears to be mediated by specific COX-2-related mechanisms. Besides, the increased intrahepatic vascular resistance in steatosis appears not to be NO-related but rather to vasoconstrictor hyperreactivity.In this study, systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis were investigated. Portal hypertension and signs of a hyperdynamic circulation were demonstrated. NO-independent, COX-2 mediated extrahepatic arterial hyporeactivity, as well as NO-independent alpha-1-adrenergic and endotheline-1 mediated intrahepatic vascular hyperreactivity are demonstrated, likely contributing to the observed portal hypertension in steatosis.

Fructose 2,6-Bisphosphate in Cancer Cell Metabolism.

For a long time, pioneers in the field of cancer cell metabolism, such as Otto Warburg, have focused on the idea that tumor cells maintain high glycolytic rates even with adequate oxygen supply, in what is known as aerobic glycolysis or the Warburg effect. Recent studies have reported a more complex situation, where the tumor ecosystem plays a more critical role in cancer progression. Cancer cells display extraordinary plasticity in adapting to changes in their tumor microenvironment, developing strategies to survive and proliferate. The proliferation of cancer cells needs a high rate of energy and metabolic substrates for biosynthesis of biomolecules. These requirements are met by the metabolic reprogramming of cancer cells and others present in the tumor microenvironment, which is essential for tumor survival and spread. Metabolic reprogramming involves a complex interplay between oncogenes, tumor suppressors, growth factors and local factors in the tumor microenvironment. These factors can induce overexpression and increased activity of glycolytic isoenzymes and proteins in stromal and cancer cells which are different from those expressed in normal cells. The fructose-6-phosphate/fructose-1,6-bisphosphate cycle, catalyzed by 6-phosphofructo-1-kinase/fructose 1,6-bisphosphatase (PFK1/FBPase1) isoenzymes, plays a key role in controlling glycolytic rates. PFK1/FBpase1 activities are allosterically regulated by fructose-2,6-bisphosphate, the product of the enzymatic activity of the dual kinase/phosphatase family of enzymes: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB1-4) and TP53-induced glycolysis and apoptosis regulator (TIGAR), which show increased expression in a significant number of tumor types. In this review, the function of these isoenzymes in the regulation of metabolism, as well as the regulatory factors modulating their expression and activity in the tumor ecosystem are discussed. Targeting these isoenzymes, either directly or by inhibiting their activating factors, could be a promising approach for treating cancers.

High Doses of Drugs Extensively Metabolized by CYP3A4 Were Needed to Reach Therapeutic Concentrations in Two Patients Taking Inducers

IntroductionIn the last 20 years of clinical practice, the senior author has identified these 2 rare cases in which the patients needed extremely high doses of drugs metabolized by CYP3A4 to reach and maintain serum therapeutic concentrations. MethodsThe high metabolic ability of these 2 patients was demonstrated by the low concentration-to-dose ratios (C/D ratios) of several drugs metabolized by CYP3A4. ResultsCase 1 was characterized by a history of high carbamazepine doses (up to 2,000mg/day) and needed 170 mg/day of diazepam in 2 days to cooperate with dental cleaning. The high activity of the CYP3A4 isoenzyme was manifested by fast metabolism for quetiapine and diazepam, which took more than 1 year to normalize after the inducer, phenytoin, was stopped. Case 2 was also very sensitive to CYP3A4 inducers as indicated by very low C/D ratios for carbamazepine, risperidone and paliperidone. The carbamazepine (2,800 mg/day) and risperidone (20 mg/day) dosages for this second patient are the highest doses ever seen for these drugs by the senior author. Risperidone induction appeared to last for many months and metabolism was definitively normal 3 years after stopping carbamazepine. On the other hand, olanzapine C/D ratios were normal for induction. ConclusionsThe literature has never described similar cases of very high doses of drugs metabolized by CYP3A4. We speculate that these 2 patients may have unusual genetic profiles at the nuclear receptor levels; these receptors regulate induction of drugs.

Whole cell melanoma vaccine genetically modified to stem cells like phenotype generates specific immune responses to ALDH1A1 and long-term survival in advanced melanoma patients

ABSTRACT Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11–19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A188-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8+ T cells. Re-stimulation with ALDH1A188-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8+ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8+ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients.

Comparative analysis of male sterility associated ATPase isoenzymes and atpA genes in a Welsh onion (Allium fistulosum L.) cytoplasmic male sterility line and its maintainer line

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the inability to produce functional pollen and is important for exploiting heterosis in crop plants, but the molecular mechanisms of CMS and fertility restoration remain poorly understood in Welsh onion. Some CMS-associated genes were previously identified by RNA-Seq as candidate genes mediating male sterility in Welsh onion. In this study, the ATPase isoenzyme and ATPase activity were investigated. We also performed 2-dimensional electrophoresis to screen for ATPase-related proteins. The ATP synthase subunit alpha was found to be specifically expressed in the CMS line. Furthermore, we found that the full length of the atpA coding sequence was the same length in the CMS line and its maintainer line, but single nucleotide polymorphisms (SNPs) that lead to amino acid substitutions in the translated proteins were found. We hypothesize that the atpA gene is involved in regulating the development of CMS.

Acetylsalicylic acid and clopidogrel hyporesponsiveness following acute coronary syndromes.

This review discusses the response variability to acetylsalicylic acid (ASA) and particularly to clopidogrel, and their relation to adverse recurrent ischaemic events in patients with arterial diseases. The higher rate of ASA resistance reported in the literature may be mainly due to the cyclooxygenase-1 non-specific assays, non-compliance, and underdosing. Clopidogrel response variability and non-responsiveness are established concepts. Moreover, high platelet reactivity (HPR) to adenosine diphosphate during clopidogrel therapy is now a known risk factor for recurrent ischaemic events in high-risk percutaneous coronary intervention/acute coronary syndrome patients. Variable active metabolite generation is the primary explanation for clopidogrel response variability and non-responsivenes. Variable levels of active metabolite generation following clopidogrel administration could be mainly explained by functional variability in hepatic cytochrome (CYP)P450 isoenzyme activity that is influenced by drug-drug interactions and single nucleotide polymorphisms of specific genes encoding CYP450 isoenzymes. Treatment with more potent P2Y12 receptor blockers, such as prasugrel and ticagrelor are credible alternative strategies to overcome HPR during clopidogrel therapy.

EFFECTS OF EXERCISE COMBINED WITH APELIN-13 ON CARDIAC FUNCTION IN THE ISOLATED RAT HEART

ABSTRACT Exercise and apelin have been shown to increase cardiac function and elicit tolerance to ischemia/reperfusion (IR) injuries. This study aimed at determining whether the combination of exercise training and apelin pretreatment could integrate the protective effects of each of them in the heart against IR injury. Male rats were divided into four experimental groups: 1: Rats with ischemia/reperfusion (IR), 2: subjected to exercise training for 8 weeks (EX+IR), 3: apelin-13 (10 nmol/kg/day) for 7 days (Apel+IR) in the last week of training, and 4: exercise training plus apelin-13 (EX+Apel+IR). Isolated hearts were perfused using the Langendorff method and subjected to 30 min of regional ischemia followed by 60 min of reperfusion. Treadmill exercise training was conducted for 8 weeks. Hemodynamic parameters were recorded throughout the experiment. Ischemia-induced arrhythmias, myocardial infarct size (IS), creatine kinase-MB (CK-MB) isoenzyme and plasma lactate dehydrogenase (LDH) activity was measured in all animals. Administration of apelin-13 plus exercise increased left ventricular developed pressure (LVDP) at the end of ischemia and reperfusion compared with other groups. After 30 min of ischemia, dP/dtmax was higher in EX+Apel+IR than in Apel+IR and EX+IR groups. During 30 min ischemia, exercise training, apelin-13 and combined treatment produced a significant reduction in the numbers of premature ventricular complexes. A combination of exercise and apelin-13 also reduced infarct size, CK-MB, LDH and severity of arrhythmia. These results suggest that combined therapies with apelin-13 and exercise training may integrate the beneficial effects of each of them alone on cardiac contractility, arrhythmia and limiting of infarct size. Level of evidence I; Therapeutic Studies - Investigating the Results of Treatment.

Effects of novel HDAC inhibitors on urothelial carcinoma cells

BackgroundHistone deacetylase inhibitors (HDACi) are promising anti-cancer drugs that could also be employed for urothelial carcinoma (UC) therapy. It is unclear, however, whether inhibition of all 11 zinc-dependent HDACs or of individual enzymes is more efficacious and specific. Here, we investigated the novel HDACi 19i (LMK235) with presumed preferential activity against class IIA HDAC4/5 in comparison to the pan-HDACi vorinostat (SAHA) and the HDAC4-specific HDACi TMP269 in UC cell lines with basal expression of HDAC4 and characterized two HDAC4-overexpressing UC cell lines.MethodsCytotoxic concentrations 50% (CC50s) for HDACi were determined by MTT assay and high-content analysis-based fluorescent live/dead assay in UC cell lines with different expression of HDAC4 and as well as in normal urothelial cell cultures, HBLAK and HEK-293 cell lines. Effects of HDACis were analyzed by flow cytometry; molecular changes were followed by qRT-PCR and Western blots. UC lines overexpressing HDAC4 were established by lentiviral transduction. Inhibitor activity profiles of HDACi were obtained by current state in vitro assays, and docking analysis was performed using an updated crystal structure of HDAC4.ResultsIn UC cell lines, 19i CC50s ranged around 1 μM; control lines were similarly or less sensitive. Like SAHA, 19i increased the G2/M-fraction, disturbed mitosis, and elicited apoptosis or in some cells senescence. Thymidylate synthase expression was diminished, and p21CIP1 was induced; global histone acetylation and α-tubulin acetylation also increased. In most cell lines, 19i as well as SAHA induced HDAC5 and HDAC4 mRNAs while rather repressing HDAC7. UC cell lines overexpressing HDAC4 were not significantly less sensitive to 19i. Reevaluation of the in vitro HDAC isoenzyme activity inhibition profile of 19i and its docking to HDAC4 using current assays suggested rather low activity against class IIA HDACs. The specific class IIA HDAC inhibitor TMP269 impeded proliferation of UC cell lines only at concentrations > 10 μM.ConclusionsAnti-neoplastic effects of 19i on UC cells appear to be exerted by targeting class I HDACs. In fact, HDAC4 may rather impede UC growth. Our results suggest that targeting of class IIA HDACs 4/5 may not be optimal for UC therapy. Moreover, our investigation provides further evidence for cross-regulation of class IIA HDACs by class I HDACs.

Screening of Barley Resistance Against Powdery Mildew by Simultaneous High-Throughput Enzyme Activity Signature Profiling and Multispectral Imaging.

Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of H. vulgare (susceptible, mildew locus o (mlo) and Mildew locus a (Mla) resistant). Multispectral imaging of barley plants was daily conducted 0–8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with mlo and Mla based resistances 0–120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of Blumeria graminis f.sp. hordei (Bgh) conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The Mla resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening.

Antioxidant Enzyme Activities in Rabbits Under Oxidative Stress Induced By High Fat Diet.

IntroductionThe aim of this study was to investigate whether the type and form of oil (raw/non-oxidised (N) or post-frying/oxidised (O)) consumed in high-fat diets affect the oxidative status of an organism, as observed by malondialdehyde (MDA) concentration as an oxidative factor and antioxidant enzyme activity.Material and MethodsFats in the diet came from rapeseed oil (R) and olive oil (O).ResultsThe applied diet caused a decrease in MDA concentration (μmol/L) in serum in group RN from 2.94 ± 0.87 to 1.76 ± 0.13, in group ON from 2.45 ± 0.62 to 1.50 ± 0.10, and in group OO from 2.70 ± 1.16 to 1.84 ± 0.36. Meanwhile, MDA concentration (mmol/L) increased in blood haemolysate in group RO from 0.15 ± 0.07 to 0.22 ± 0.03 and in group OO from 0.17 ± 0.02 to 0.22 ± 0.02. The observed changes caused a response of the enzymatic antioxidant system in both models, especially followed by an increase in activities of total superoxide dismutase and its mitochondrial isoenzyme in all experimental groups, while its cytosolic isoenzyme activity increased only in ON and OO groups. Increased activity of glutathione peroxidase (GPX) in groups RN and RO and of catalase (CAT) in groups ON and OO was observed. Significant differences in responses to the different types and forms of oils were probably caused by the different oxidative stability of the studied oils.ConclusionThis diet disturbed the body’s oxidative status; however, during the six-month study the enzymatic antioxidant system remained effective.

Purification and biochemical characterization of peroxidase isoenzymes from Ficus carica latex

Abstract Three peroxidase isoenzymes were isolated from Ficus carica latex using CM-Sepharose, DEAE-Sepharose and Sephacryl S-200. The complete purification was carried out for FP1 only due to the low level of activity and protein concentration of FP2 and FP3. The purified isoenzyme FP1 was found to be monomeric with a molecular weight of 30 kDa. FP1 and FP3 isoenzymes had the same pH and temperature optima at pH 5.5 and 40 °C, whereas the optimum values of pH and temperature were at pH 7.0 and 30 °C for FP2. On the other hand, FP1, FP2 and FP3 were stable at 50 °C, 40 °C and 30 °C respectively, whereas FP3 had low thermostability. FP1 isoenzyme was found to be stable between pH 5.0 and 7.5, and FP2 was stable from pH 4.0–8.0, while FP3 was found to be stable in acidic range between pH 4.5 and pH 5.5. The activity of both FP1 and FP2 peroxidase isoenzymes was increased by the high concentration of Ca2+ (10 mM). The three peroxidase isoenzymes have a broad specificity towards some phenolic substrates and O -Phenylenediamine showed higher affinity towards the three peroxidase isoenzymes, with Km values of 3.87, 3.64 and 3.33 mM for FP1, FP2 and FP3. The properties of these isoenzymes meet the prerequisites needed for environmental and industrial applications.

PHARMACOKINETIC AND PHARMACOGENETIC ASPECTS OF PERSONALIZED ANALGETIC THERAPY WITH FENTANIL TTS IN CLINICAL ONCOLOGY

The aim of the study was the pharmacokinetic and pharmacogenetic analysis of analgetic efficacy and safety of transdermal fentanyl for cancer patients.Material and methods. A comprehensive search for journal articlespublished between 2012 and 2017 was carried out using PubMed, Scopus, Web of Science, and E-library databases.Results. The analysis of the data showed that pharmacokinetic and pharmacogenetic factors can influence the interindividual variability of analgesic therapy with fentanyl TTC for cancer patients, predetermining phenotypic differences in the efficacy and safety of analgesia. Enforced polypharmacotherapy with the use of inducers or inhibitors of the CYP3A4 isoenzyme activity can significantly change the effectiveness of analgesic therapy and result in undesirable side effects of strong opioids. Contradictory data on the effect of some single nucleotide polymorphisms of metabolic genes, transport genes and mu-opioid receptor genes dictate the necessity of further studies in this field.Conclusion. To date, there is no single explanation for interindividual variability of analgesic therapy with fentanyl TTS. A comprehensive assessment of the pharmacokinetic and pharmacogenetic factors affecting the efficacy and safety of analgesic therapy with potent opioids is a tool of a personalized approach for anesthesia in clinical oncology.

Methods for Determination of Functional Activity of Cytochrome P450 Isoenzymes

The review deals with modern methods and technologies for determination of functional activity cytochrome P450 isoenzymes; these include absorbance and fluorescent spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Raman, Mossbauer, and X-ray spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM). Methods of molecular genetic analysis have been considered in the context of personalized medicine. Using the methods of chromatography mass spectrometry it is possible to analyze reaction products formed in reactions catalyzed by cytochromes P450. Special attention is paid to modern electrochemical systems based on cytochrome P450 isoenzymes, their applicability for analysis of their catalytic activity, their use in practice and further development perspectives for experimental pharmacology, biotechnology and translational medicine.

Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in the Serum of Patients with Non-alcoholic Fatty Liver Disease.

Non-alcoholic liver disease (NAFLD) is one of the most common causes of chronic liver disease, and its prevalence and medical importance is increasing worldwide. Changes in enzyme activity in liver cells in various liver diseases are reflected by an increase in serum enzymatic activity. For example, alcohol dehydrogenase activity (ADH) and aldehyde dehydrogenase (ALDH), that occur in the liver in large quantities, correlate with disease severity during cirrhosis. In the current study, the activity of ADH isoenzymes and ALDH in the serum of patients with NAFLD was investigated. Serum samples were collected for routine biochemical studies from 55 patients with NAFLD patients and from 50 healthy individuals. Class I and II ADH and ALDH activity were measured by spectrofluorometric method. Photometric methods were used to measure ADH class III, IV and total ADH activity. Total ADH activity was significantly higher in non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) than in healthy individuals (44 and 48.5% activity, respectively). The median total activity of ADH was 1,164 mU/l in patients with NAFLD, 1,258 mU/l in NASH and 648 mU/l in the control group. The increase in ADH class I and II isoenzyme in serum of patients with NAFL and NASH was statistically significant. The activity of ADH I, ADH II, and total ADH significantly increased with increasing disease progression. The activity of isozymes of class I and II alcohol dehydrogenase in patients with NAFLD is enhanced and appears to be due to the release of these isoenzymes from damaged hepatocytes.

Regulation of the key antioxidant enzymes by developmental processes and environmental stresses in the dark

The presence of solar radiation is one of the most important environmental factors, which is required for the optimal growth and development of plants. The absence of it (e.g. in the night period or artificially prolonged darkness) can alter the light-dependent signalling and regulation pathways and may induce new defence responses. Antioxidant enzymes as components of the plant defence system play a crucial role in the detoxification of reactive oxygen species (ROS) induced by several stressors; however, their regulation can be different in the light or in the dark. In this review we summarize the current knowledge about the physiological and molecular aspects of dark-modulated key antioxidant enzymes (superoxide dismutase, catalase, and ascorbate peroxidase) in different plant species and discuss their roles in different developmental processes (seedling growth and development or senescence) and in responses to environmental stresses (cold, chilling, heat, and biotic stress). Moreover, the hormonal regulation of respective gene transcription and the changes in activity of various isoenzymes at subcellular level are also summarized. Based on this knowledge, modification of these antioxidant enzymes may be used to increase the yield and stress tolerance of cultivated plants in the changing environment.

Antioxidant responses and isoenzyme activity of hydroponically grown safflower seedlings under copper stress

Safflower is an oilseed rabi crop. Continuous use of use of copper (Cu) containing fungicides and pesticides, due to high susceptibility of this crop of fungal attack has increased the accumulation of Cu in the soil. To investigate the effect of extra accumulated Cu on this particular crop, safflower seeds were germinated on different concentration of Cu (25, 50 and 100 µM) present in Hoagland’s solution (hydroponic) along with control (0.5 µM of Cu) and seedlings were harvested after 10 and 20 days of Cu treatment. The concentration of alpha-tocopherol was high in leaves as compared to roots in presence of Cu stress. The content of vitamin C was increased in both leaves and roots. In 10 and 20 day leaves, the CAT activity has been decreased in all the treatments. In 10 day roots, the CAT activity was increased but decreased abruptly in 20 day harvested roots. Isoenzymatic pattern of CAT showed three bands of CAT (CAT 1, CAT 2 and CAT 3) in control in 10 day leaves while CAT 1 was diminished in lane 2, 3 and 4. In 20 day leaves, we observed only one bands of CAT (CAT 2) in lane 1 (control) while CAT 1 was appeared at high concentration of Cu. We observed no more significant changes in the CAT isoforms in 10 and 20 day roots. The GPX activity was increased in both the day’s collected samples (leaves and roots). The analysis of GPX isoenzymes revealed no significant changes in isoenzymatic pattern but their band’s intensity was increased with an elevated metal treatment. In 20 day harvested leaves, we observed two bands of isoenzyme in lane 1 and four bands of GPX in lane 2, 3, 4 along with their increased intensities. In 10 day roots, we observed two band of GPX in all the treatment with elevated intensities as compared to control (lane 1). Similarly we observed two bands of GPX in all the treatments and control. But the intensity of bands was increased till lane 3 but in 20 day rootsat highest treatment (100 µM) the intensity of bands was decreased. The SOD activity was reduced in 10 and 20 day leaves. In 10 day roots, it was augmented but reduced at 100 µM as compared to control (100%). In 20 day harvested roots, the activity was decreased by 1.30, 11.11 and 15.68% with increased Cu concentration than control (100%). Three isoenzymes of SOD were appeared in 10 day leaves with more or less same intensity. While in 20 day leaves ex-plant has shown SOD 1, 2 and 3 activities. In 10 day treated roots SOD 1 with increasing intensity was appeared in lane 3 and 4 than control (lane 1) while SOD 2 was almost constantly present in all four lanes. Similarly in 20 day root ex-plant, SOD 1 has been observed with decreasing intensity in lane 2, 3 and 4 than control (lane 1). To cope up with the Cu stress, the antioxidant enzymes such as GPX and SOD were actively involved in safflower seedlings.