Isoenzyme Activity Research Articles

Лабораторные критерии мониторинга патологического процесса у больных с хроническим остеомиелитом голени на этапах восстановительного лечения

Purpose Search for laboratory criteria of pathological process monitoring in patients with chronic osteomyelitis of the tibia at the stages of its management using the method of transosseous distraction osteosynthesis. Materials and methods Biochemical parameters of blood serum were studied in 15 patients with chronic posttraumatic osteomyelitis of the tibia. Sequester necrectomy with subsequent osteosynthesis of the tibia with the Ilizarov apparatus was performed in all patients at the first stage. At the second stage, the defect was filled in with distraction regenerate according to Ilizarov. Depending on the bone defect size, the patients were divided into two groups. The defect size was from 3 to 5 cm in patients of the first group (n = 7, mean age 42.4 ± 6.9 years). In the patients of the second group (n = 8, mean age 37.5 ± 7.8 years) the size of the defect was from 7 to 12 cm. Results It was found that the changes in the activity of the bone isoenzyme of acid phosphatase (TRAP), alkaline phosphatase, creatine phosphokinase, lactate and C-reactive protein (CRP) did not differ significantly between the groups during their treatment. At the same time, the concentration of CRP, sialic acids and the activity of TRAP in the first stage perids of treatment was significantly increased in patients of both groups relative to the reference norm. In patients of the second group, the concentration of sialic acids was higher than the values of group 1 and the reference group in the periods of 7-21 days of fixation. Nevertheless, at the beginning of the second stage of treatment (distraction), the level of all the studied parameters was not significantly different from the norm. Conclusion Normalization of TRAP activity, concentration of CRP and sialic acids values can be attributed to additional diagnostic features on the basis of which it is possible to start the second stage of treatment (distraction) in patients with chronic osteomyelitis of the tibia.

Do CYP2C19 and ABCB1 gene polymorphisms and low CYP3A4 isoenzyme activity have an impact on stent implantation complications in acute coronary syndrome patients?

AimThe aim of this study was to determine the impact of CYP2C19 and ABCB1 gene polymorphisms and CYP3A4 isoenzyme activity on stent implantation complications among patients with an acute coronary syndrome (ACS) who underwent percutaneous coronary intervention (PCI).Patients and methodsSeventy-six patients (median age 63, range 37–91 years) with an ACS who underwent PCI were screened for CYP2C19 and ABCB1 gene polymorphisms with real-time polymerase chain reaction: CYP2C19*2, CYP2C19*17, and ABCB1 3435. CYP3A4 isoenzyme activity was determined by urine cortisol and 6-beta-hydroxycortisol levels. Stent implantation complications such as stent thrombosis (n=2) and restenosis (n=1) were observed among drug-eluting stent recipients.ResultsLow mean 6-beta-hydroxycortisol/cortisol ratio is indicative of impaired CYP3A4 activity and was associated with higher risk of thrombosis (b coefficient=0.022, SE 0.009, p=0.021 in the linear regression model). The increase in the length of the implanted stent was associated with higher risk of restenosis (b coefficient=0.006, SE=0.002, p=0.001 in the linear regression model). The presence of the CYP2C19*2 polymorphism did not affect the incidence of stent thrombosis (b coefficient=−1.626, SE=1.449, p=0.262 in the logistic regression model), nor did the CYP2C19*17 (b coefficient=−0.907, SE=1.438, p=0.528 in the logistic regression model) and ABCB1 3435 polymorphisms (b coefficient=1.270, SE=1.442, p=0.378 in the logistic regression model).ConclusionWe did not find evidence that the presence of CYP2C19*2, CYP2C19*17, and ABCB1 3435 polymorphisms may jeopardize the safety of stent implantation in patients with an ACS. Patients with low CYP3A4 isoenzyme activity may have increased risk of stent thrombosis.

The inhibition of inducible nitric oxide synthase and oxidative stress by agmatine attenuates vascular dysfunction in rat acute endotoxemic model

Vascular dysfunction leading to hypotension is a major complication in patients with septic shock. Inducible nitric oxide synthase (iNOS) together with oxidative stress play an important role in development of vascular dysfunction in sepsis. Searching for an endogenous, safe and yet effective remedy was the chief goal for this study. The current study investigated the effect of agmatine (AGM), an endogenous metabolite of l-arginine, on sepsis-induced vascular dysfunction induced by lipopolysaccharides (LPS) in rats. AGM pretreatment (10mg/kg, i.v.) 1h before LPS (5mg/kg, i.v.) prevented the LPS-induced mortality and elevations in serum creatine kinase-MB isoenzyme (CK-MB) activity, lactate dehydrogenase (LDH) activity, C-reactive protein (CRP) level and total nitrite/nitrate (NOx) level after 24h from LPS injection. The elevation in aortic lipid peroxidation illustrated by increased malondialdehyde (MDA) content and the decrease in aortic glutathione (GSH) and superoxide dismutase (SOD) were also ameliorated by AGM. Additionally, AGM prevented LPS-induced elevation in mRNA expression of iNOS, while endothelial NOS (eNOS) mRNA was not affected. Furthermore AGM prevented the impaired aortic contraction to KCl and phenylephrine (PE) and endothelium-dependent relaxation to acetylcholine (ACh) without affecting endothelium-independent relaxation to sodium nitroprusside (SNP). In conclusion: AGM may represent a potential endogenous therapeutic candidate for sepsis-induced vascular dysfunction through its inhibiting effect on iNOS expression and oxidative stress.

Increasing creatine kinase activity protects against hypoxia / reoxygenation injury but not against anthracycline toxicity in vitro.

The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P<0.05). Loading cells with creatine did not modify cell survival. Transient expression of CK isoforms in HL-1 cardiac cells elevated isoenzyme activity, but only CKMT2 over-expression protected against hypoxia (0.1% for 24h) and reoxygenation demonstrating 25% less cell death compared to non-transfected control (P<0.01). The same cells were not protected from doxorubicin toxicity (250nM for 48h), in contrast to the positive control. These findings support increased CK activity as protection against ischaemia-reperfusion injury, in particular, protection via CKMT2 in a cardiac-relevant cell line, which merits further investigation in vivo.

The alterations in alcohol dehydrogenase and aldehyde dehydrogenase activities in the sera of patients with renal cell carcinoma

PurposeIn a previous study we showed that the total activity of alcohol dehydrogenase (ADH) and its isoenzyme class I was significantly higher in renal cancer (RCC) cells compared to normal kidney. The aim of this study was to compare the activities of ADH isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with different stages of RCC and healthy subjects. Materials and methodsSerum samples were taken from 54 patients with clear cell RCC (17 patients in stage II, 22 in stage III and 15 in stage IV) and 52 healthy patients. Class III, IV of ADH and the total ADH activity was measured by the photometric method. For the measurement of ADH class I, II and the total ALDH activity we employed the fluorometric method. ResultsThe total activity of ADH and its isoenzyme class I were significantly higher in the sera of patients with every stage of RCC compared to healthy subjects. The analysis of ALDH activity did not indicate significant differences between tested groups. ConclusionsThe increased activity of total ADH and its isoenzyme class I in the sera of patients with RCC, seems to be caused by isoenzymes being released from cancerous cells and may be useful for diagnostics of renal cancer.

Критерії вибору методу дослідження

Критерії вибору методу дослідження

Assessment of CYP2C19, ABCB1, CYP3A5 Genes Polymorphisms’ and CYP3A4 Isoenzyme Activity Influence on Major Adverse Cardiovascular Events Among Patients with an Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention

Assessment of CYP2C19, ABCB1, CYP3A5 Genes Polymorphisms’ and CYP3A4 Isoenzyme Activity Influence on Major Adverse Cardiovascular Events Among Patients with an Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention

222 Augmentation of creatine kinase in vitro protects against simulated ischaemia reperfusion injury

Creatine kinase (CK) catalyses the interchange of high energy phosphates to buffer ATP levels and maintains cellular energy homeostasis. The heart expresses three isoforms: sarcomeric mitochondrial CK (CKMT2), and the...

Synthesis and carbonic anhydrase inhibitory properties of new spiroindoline-substituted sulphonamide compounds

New spiroindoline-substituted sulphonamide compounds were synthesised 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 14 synthesised sulphonamides (6a–n) on esterase activities of these isoenzymes were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesised compounds inhibited the carbonic anhydrase (CA) isoenzyme activity. Among them, 6b was found to be the most active (Ki: 0.042 μM) for hCA I and 6a (Ki: 0.151 μM) for hCA II.

Investigation on the effects of pomiferin from Maclura pomifera on indomethacin-induced gastric ulcer: An experimental study in rats

Previously, water extract of Maclura pomifera plant has been found effective on gastric damage. In this study, pomiferin product was purified from this plant by thin-layer chromatography method and its structure was elucidated by nuclear magnetic resonance methods. Its antiulcer effect was investigated on indomethacin-induced gastric damage in rats. For this study, a total of 36 Sprague Dawley female rats were divided into six groups such as healthy, control, reference, and three treatment groups (n = 6 each). Ranitidine (Ulcuran® 25 mg/ml vial) was used as reference. Biochemically, lipid peroxidation, and glutathione levels as well as superoxide dismutase, catalase, myeloperoxidase, and cyclooxygenase isoenzymes activities were measured in stomach tissues. Comparing with healthy group, ulcerative area was observed in control group (only administered to indomethacin group). However, Ulcerative area in stomach tissues of rats administered of all doses of pomiferin and ranitidine showed significant decrease (p < 0.05). Also, there were significant changes at the antioxidant levels (lipid peroxidation, glutathione, superoxide dismutase, and catalase) and some important enzyme activities (myeloperoxidase, cyclooxygenase isoenzymes). We found that pomiferin was effective on gastric damage induced by indomethacin. This effect was thought to be caused by positive changes in the antioxidant system.

Mercury induced oxidative stress, DNA damage, and activation of antioxidative system and Hsp70 induction in duckweed (Lemna minor)

Mercury uptake and its effects on physiology, biochemistry and genomic stability were investigated in Lemna minor after 2 and 6d of exposure to 0–30μM Hg. The accumulation of Hg increased in a concentration- and duration-dependent manner, and was positively correlated with the leaf damage. Oxidative stress after Hg exposure was evidenced in L. minor by a significant decrease in photosynthetic pigments, an increase in malondialdehyde and lipoxygenase activities (total enzyme activity and isoenzymes activity). Fronds of L. minor exposed to Hg showed an induction of peroxidase, catalase, and ascorbate peroxidase activities (total enzyme activity and some isoenzymes activities). Exposure of L. minor to Hg reduced the activity (total enzyme activity and some isoenzymes activities) of glutathione reductase, and superoxide dismutase. Exposure to Hg produced a transient increase in the content of glutathione and ascorbic acid. The content of dehydroascorbate and oxidized glutathione in L. minor were high during the entire exposure period. Exposure of L. minor to Hg also caused the accumulation of proline and soluble sugars. The amplification of new bands and the absence of normal DNA amplicons in treated plants in the random amplified polymorphic DNA (RAPD) profile indicated that genomic template stability (GTS) was affected by Hg treatment. The accumulation of Hsp70 indicated the occurrence of a heat shock response at all Hg concentrations. These results suggest that L. minor plants were able to cope with Hg toxicity through the activation of various mechanisms involving enzymatic and non-enzymatic antioxidants, up-regulation of proline, and induction of Hsp70.

Personalized medicine in internal medicine

Personalized medicine - a new direction in medicine, which is based on the study of various biomarkers and the use of new methods of molecular analysis (primarily evaluating the activity of isoenzymes of cytochrome P450), allowing individualized approach to the selection of both the drugs and the selection of the dosing regimen for the purpose of maximize the effectiveness and safety of pharmacotherapy. This personalized medicine is to change the development and use of preventive and curative interventions. Genetic polymorphism isozymes of cytochrome P450 may determine the individual activity of a particular isozyme, and thus, to predict the clinical effectiveness, and in some cases, the risk of adverse reactions. The article is an example of the use of information on the activity of cytochrome P450 in clinical practice in matters of diagnosis, treatment and prevention of diseases. The scheme of the five best-known activity of isoenzymes of cytochrome P450 is shown.

Serum Creatine Kinase-MB Isoenzyme Activity among Subjects with Uncomplicated Essential Hypertension: Any Sex Differences.

Hypertension (high blood pressure) is a major health challenge and more women than men are affected by the condition. Complications as a result of this condition often lead to disabilities and premature death. The objective of this study was to evaluate creatine kinase-MB (CK-MB) activity in uncomplicated hypertension and to know whether sex differences exist in the activity of the enzyme. Serum creatine kinase-MB isoenzyme activity, troponin I, and lipid profile were evaluated in 140 male and 100 female Nigerians with hypertension. The control group was comprised of 100 (50 males and 50 females) normotensive subjects. Measured parameters were assayed using Selectra Pros chemistry analyzer. The means were compared between males and females using Students’t-test. The mean CK-MB activity of the female hypertensive subjects was significantly higher (p < 0.001) than the males. Similarly, the mean cardiac troponin I (cTnI) of the female hypertensive subjects was significantly higher (p < 0.001) than the males. Conversely, the mean CK-MB activity of the female normotensive subjects was significantly lower (p < 0.001) than the male counterparts. There was no difference in the levels of cTnI between male and female normotensive subjects. Serum CK-MB activity was higher in female than male hypertensive subjects. In the light of these results, cardiac markers should be routinely done in the evaluation of hypertensive subjects and sex-specific consideration may be recognized in the management of these patients.

Protease prospecion and determination of its isoenzymes activity in cocoa cultivars (Theobroma cacao L.)

Our objective was to characterize the protease enzymatic activity and its isoenzymes on cacao cultivars PH 16 and TSH 1188, produced in southern Bahia, linking it to the conditions of the fermentation process. Proteases were extracted and semi-purified, their activities determined changing substrate, pH and temperature, and the values ​​compared with the parameters of fermentation (pH and temperature), and yet determined the kinetic parameters and the activity of its isoenzymes: aminopeptidase, carboxypeptidase and endoprotease. In the experimental conditions, differences in protease activities were shown as to different cocoa cultivars on various conditions. The albumin stands out as the preferred enzyme substrate, in a pH range between 3 and 6 and temperatures between 29 ° C and 50 ° C. As for the isoenzymes activity, an increased activity in these seeds and in the cultivar TSH 1188 was observed. When correlated with the fermentative parameters, the conditions for enzymatic activity are not the best determined, with emphasis on farming pH 16 presenting its fermentation conditions far from those found as optimal, especially in pH evaluation, since temperature varies very little between the two cultivars.

Evaluation of lactate dehydrogenase isoenzyme activity and biochemical profile in cerebrospinal fluid for different types of meningitis

Evaluation of lactate dehydrogenase isoenzyme activity and biochemical profile in cerebrospinal fluid for different types of meningitis

Anisucoumaramide, a Bioactive Coumarin from Clausena anisum-olens.

A new coumarin, anisucoumaramide (1), and a new δ-truxinate derivative, anisumic acid (2), were isolated from Clausena anisum-olens. Their structures were elucidated from extensive NMR and MS data. The absolute configurations of the coumarins were assigned using the experimental and calculated electronic circular dichroism data. Anisucoumaramide (1) represents the first example of a naturally occurring coumarin of which the terpenoidal side chain does not comply with the biosynthesis isoprene rule due to the presence of an unprecedented acetamido motif directly connected with the terpenoidal side chain. The δ-truxinate derivative was isolated from Clausena species for the first time. Compound 1 showed high selectivity for the MAO-B isoenzyme and inhibitory activity in the nanomolar range. Putative biosynthesis pathways toward 1 and 2 are proposed.

The impact of porous silicon nanoparticles on human cytochrome P450 metabolism in human liver microsomes in vitro

Engineered nanoparticles are increasingly used as drug carriers in pharmaceutical formulations. This study focuses on the hitherto unaddressed impact of porous silicon (PSi) nanoparticles on human cytochrome P450 (CYP) metabolism, which is the major detoxification route of most pharmaceuticals and other xenobiotics. Three different surface chemistries, including thermally carbonized PSi (TCPSi), aminopropylsilane-modified TCPSi (APTES-TCPSi) and alkyne-terminated thermally hydrocarbonized PSi (Alkyne-THCPSi), were compared for their effects on the enzyme kinetics of the major CYP isoforms (CYP1A2, CYP2A6, CYP2D6, and CYP3A4) in human liver microsomes (HLM) in vitro. The enzyme kinetic parameters, Km and Vmax, and the intrinsic clearance (CLint) were determined using FDA-recommended, isoenzyme-specific model reactions with and without PSi nanoparticles. Data revealed statistically significant alterations of most isoenzyme activities in HLM in the presence of nanoparticles at 1mg/ml concentration, and polymorphic CYP2D6 was the most vulnerable to enzyme inhibition. However, the observed CYP2D6 inhibition was shown to be dose-dependent in case of TCPSi and Alkyne-THCPSi nanoparticles and attenuated at the concentrations below 1μg/ml. Adsorption of the probe substrates onto the hydrophobic Alkyne-THCPSi particles was also observed and taken into account in the determination of the kinetic parameters. Three polymer additives commonly used in pharmaceutical nanoformulations (Pluronics F68 and F127, and polyvinylalcohol) were also separately screened for their effects on CYP isoenzyme activities. These polymers had less effect on the enzyme kinetic parameters, and resulted in increased activity rather than enzyme inhibition, in contrast to the PSi nanoparticles. Although the chosen subcellular model (HLM) is not able to predict the cellular disposition of PSi nanoparticles in hepatocytes and thus provides limited information of probability of CYP interactions in vivo, the present study suggests that mechanistic interactions by the PSi nanoparticles or the polymer stabilizers may appear if these are effectively uptaken by the hepatocytes.

Metabolic Interference of sod gene mutations on catalase activity in Escherichia coli exposed to Gramoxone® (paraquat) herbicide

Herbicides are continuously used to minimize the loss of crop productivity in agricultural environments. They can, however, cause damage by inhibiting the growth of microbiota via oxidative stress, due to the increased production of reactive oxygen species (ROS). Cellular responses to ROS involve the action of enzymes, including superoxide dismutase (SOD) and catalase (CAT). The objective of this study was to evaluate adaptive responses in Escherichia coli K-12 to paraquat, the active ingredient in the herbicide Gramoxone®. Mutant bacterial strains carrying deletions in genes encoding Mn-SOD (sodA) and Fe-SOD (sodB) were used and resulted in distinct levels of hydrogen peroxide production, interference in malondialdehyde, and viability. Mutations also resulted in different levels of interference with the activity of CAT isoenzymes and in the inactivation of Cu/Zn-SOD activity. These mutations may be responsible for metabolic differences among the evaluated strains, resulting in different patterns of antioxidative responses, depending on mutation background. While damage to the ΔsodB strain was minor at late log phase, the reverse was true at mid log phase for the ΔsodA strain. These results demonstrate the important role of these genes in defense against oxidative stress in different periods of growth. Furthermore, the lack of Cu/Zn-SOD activity in both mutant strains indicated that common metal cofactors likely interfere in SOD activity regulation. These results also indicate that E. coli K-12, a classical non-environmental strain, constitutes a model of phenotypic plasticity for adaptation to a redox-cycling herbicide through redundancy of different isoforms of SOD and CAT enzymes.

Alpha-Tocopherol-Dependent Salt Tolerance is More Related with Auxin Synthesis Rather Than Enhancement Antioxidant Defense in Soybean Roots

In this paper, we describe the alleviated effects of alpha-tocopherol (α-T) on oxidative damage and its possible role as a signal transmitter in plants during salt stress. The results show that exogenously applied α-T under salt stress increased root length and weight, but reduced hydrogen peroxide (H2 O2), superoxide anion radical (O2.–) and malondialdehyde (MDA) content in soybean roots. The proline content was reduced by α-T treatment. Interestingly, endogenous auxin (IAA) level was significantly increased after α-T application as compared to salt stress alone. Moreover, α-T reduced significantly superoxide dismutase (SOD) enzyme and isoenzyme activity but upregulated peroxidase (POX) 2, 3 and glutathione-stransferase (GST) 1, 3 isoenzyme expression. However, ascorbate peroxidase (APX) enzyme activity was not affected at all. Consequently, the results show that α-T serves as a signal molecule under salinity from leaves to roots by increasing remarkably endogenous IAA levels and increasing partially antioxidant activity in roots.

Food Consumption and Electrophoresis of Some Isoenzymes in the Motor Muscles of Schistocerca gregaria Fed on Lotus corniculatus

The desert locust, Schistocerca gregaria (Forskal, 1775) (Orthoptera, Acrididae), may feed in the Sahara desert on Lotus corniculatus (Fabaceae), which is rich in linamarin, a cyanogenic glucoside that is usually toxic for herbivores. The relationship between food consumption and the activity of five isoenzymes in the motor muscles of desert locust was investigated. Nutritional indices were estimated in fifth instar female nymphs. Consumption and assimilation rates on L. corniculatus were significantly lower than those on wheat seedlings (Triticum aestivum) as control plant. The efficiency of ingested (ECI) and digested (ECD) food conversion was significantly lower in the nymphs fed on L. corniculatus than for those fed on wheat seedlings. The relative growth rate (RGR) and relative consumption rate (RCR) for nymphs feeding on L. corniculatus were significantly lower. The development duration of nymphs fed on Lotus was relatively longer than that for those fed on wheat seedlings. Nymphs fed on L. corniculatus lost weight. Electrophoretic assays of five enzymes were assessed in adult females to examine the feeding effect on L. corniculatus and T. aestivum on the isoenzymatic patterns of these enzymes. Considered enzymes were acid phosphatase (Acph), alcohol dehydrogenase (Adh), β-esterase (β-Est), malic enzyme (Mal) and malate dehydrogenase (Mdh). Enzymes were tested in femur and wing muscles of adult females. Feeding on T. aestivum seemed to be more efficient for the activity of the metabolic enzymes (Acph, Mal and Mdh) than feeding on L. corniculatus. Their isoenzymes were relatively more expressed in the case of feeding on T. aestivum. The activity of esterase isoenzymes (β-Est) was higher in the case of feeding on L. corniculatus thus indicating the role of this particular enzyme in detoxifying plant linamarin allelochemicals.