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1
WELLER, DAVID L. "ACIDIC PEROXIDASES OF APPLE ROOTSTOCKS." Canadian Journal of Plant Science 67, no. 1 (January 1, 1987): 293–97. http://dx.doi.org/10.4141/cjps87-042.
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The peroxidase activity in extracts of shoot bark from Michigan State Apple Clone (MAC) 24 and in extracts of roots of MAC 24, East Mailing Long Ashton (EMLA) 27, and Oregon Apple Rootstock (OAR) 1 were characterized as being primarily acidic by broad range sucrose gradient isoelectric focusing. Gel isoelectric focusing (GIEF) using a narrower conventional or immobilized pH gradient showed that this activity could be separated into about a dozen isoenzymes. The GIEF isoenzyme patterns of MAC 24 shoot bark extract peroxidase and root extract peroxidases of MAC 24, EMLA 27, and OAR 1 differed. Similar values were obtained for the molecular weight of the peroxidase activity of these extracts. The results indicate that the acidic isoenzymes of apple peroxidase are of similar size.Key words: Apple, rootstocks (apple), peroxidase, molecular weight, isoenzymes
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Najafi, Mohammad, Abazar Roustazadeh, Ali Asghar Moshtaghie, and Mohsen Ani. "Liver Aspartate Transaminase Isoenzymes as Biomarkers of Chronic Exposure to Chromium(VI)." Archives of Industrial Hygiene and Toxicology 64, no. 4 (December 1, 2013): 547–52. http://dx.doi.org/10.2478/10004-1254-64-2013-2358.
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Abstract Exposure to hexavalent chromium compounds is associated with the risk of lung cancer, dermatitis, gastrointestinal ulcers, and other tissue damages. The aim of this study was to compare liver isoenzyme and total serum activities of aspartate aminotransferase (AST) as cytotoxic biomarkers of acute and chronic cytotoxicity of CrVI. We investigated the extent of cell damage caused by chromium(VI) in acute (2.5 mg kg-1) daily doses administered over five days and chronic (0.25 mg kg-1 and 0.5 mg kg-1) daily doses administered over 15 to 60 days by measuring total AST in serum and low molecular weight AST (LMW-AST) and high molecular weight AST (HMW-AST) activities in thirty liver fractions. We also evaluated the kinetic properties and electrophoretic mobility of the LMW- and HMW-AST isoenzymes in liver subcellular fractions. Liver LMW-AST and total serum AST activities significantly decreased after 15 days of exposure (P<0.05). With continued treatment, AST activity increased by 15.67 % (P<0.05). Interestingly, changes in serum AST activity were similar to changes in the liver LMW-AST isoenzyme. Our results confirmed that total serum AST activity may serve as a reliable tissue biomarker for long-term exposures to CrVI, but they also suggest that the LMW-AST isoenzyme could be even more sensitive.
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Toups, Erin A., and Enmin Zou. "Epidermal collagenase activity and its induction by 20-hydroxyecdysone in the fiddler crab Uca pugilator." Current Zoology 55, no. 1 (February 1, 2009): 75–80. http://dx.doi.org/10.1093/czoolo/55.1.75.
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Abstract The epidermal collagenase activity and its induction by 20- hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular weight, with the former two accounting for 60% and 36%, respectively, of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested, Premolt Stage D0 exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and, as the molt stage proceeded, the enzymatic activity of these two isoenzymes decreased, with the lowest activity for both found in Premolt Stage D3-4. Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa collagenase in the epidermis of Uca pugilator, suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyecdysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme, a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean.
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Trbojevic, Jovana, Aleksandra Dimitrijevic, Dusan Velickovic, Marija Gavrovic-Jankulovic, and Nenad Milosavic. "Isolation of Candida rugosa lipase isoforms." Chemical Industry 67, no. 5 (2013): 703–6. http://dx.doi.org/10.2298/hemind120828113t.
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Convenient source of lipases for science and industry is yeast Candida rugosa. Crude preparation of Candida rugosa lipase (CRL) consists of several extracellular lipases. Isoenzyme profile depends on the culture or fermentation conditions. All isoforms are coded by lip pseudogene family; they are monomers of 534 amino acids and molecular weight of about 60 kDa. They share the same catalytic mechanism and interfacial mode of activation. Isoenzymes differ in isoelectric points, post-translational modifications, substrate specificity and hydrophobicity. The presence of different lipase isoforms and other substances (i. e. inhibitors) in crude preparation leads to lack of their productivity in biocatalytic reactions. Purification of specific isoform improves its overall performance and stability. This paper provides an overview of different methods for purification of CRL isoenzymes up to date, their advantages and disadvantages.
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Bourtzis, K., and V. J. Marmaras. "Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization." Biochemistry and Cell Biology 69, no. 10-11 (October 1, 1991): 731–35. http://dx.doi.org/10.1139/o91-110.
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Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KC1, respectively. Both isoenzymes have a molecular weight of approximately 180 000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and β-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.Key words: enzyme, Diptera, integument, phosphatase, isoenzymes, phosphotyrosine.
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Caselli, Anna, Maria Letizia Taddei, Chiara Bini, Paolo Paoli, Guido Camici, Giampaolo Manao, Paolo Cirri, and Giampietro Ramponi. "Low Molecular Weight Protein Tyrosine Phosphatase and Caveolin-1: Interaction and Isoenzyme-Dependent Regulation†." Biochemistry 46, no. 21 (May 2007): 6383–92. http://dx.doi.org/10.1021/bi0620858.
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Mavrides, Charalampos, and Guylaine Nadeau. "Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart." Biochemistry and Cell Biology 65, no. 3 (March 1, 1987): 239–44. http://dx.doi.org/10.1139/o87-031.
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The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate–aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.
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Krejčová, Romana, Květoslava Horská, Ivan Votruba, and Antonín Holý. "Phosphorylation of Purine (Phosphonomethoxy)alkyl Derivatives by Mitochondrial AMP Kinase (AK2 Type) from L1210 Cells." Collection of Czechoslovak Chemical Communications 65, no. 10 (2000): 1653–68. http://dx.doi.org/10.1135/cccc20001653.
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Substrate activity of purine (phosphonomethoxy)alkyl derivatives towards mitochondrial AMP kinase (AK2 type) from L1210 cells was studied. The native AMP kinase, purified nearly to homogeneity, is a monomer with molecular weight 26 kDa. The purified AMP kinase is specific for natural adenine nucleotides (AMP and dAMP) as phosphate acceptors but has a broad specificity to nucleoside 5'-triphosphates as phosphate donors. In addition to adenine acyclic nucleotide analogues, the enzyme is capable of phosphorylating also analogous derivatives containing 2,6-diaminopurine moiety. Kinetic data show that the substrate activity of these acyclic nucleoside phosphonates towards AK2 isoenzyme decreases in the order (S)-HPMPA > (R)-PMPA > PMEA > PMEDAP > (S)-PMPDAP > (R)-PMPDAP >> (S)-PMPA. Acyclic nucleotide analogues do not exhibit any inhibitory activity towards AK2 isoenzyme.
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Al-Juraisy1, Amel Taha Yassein, Ameera Aziz Mahmood Al-Juraisy1, Ameera Aziz Mahmood Al-Juraisy1, and Nadia Ahmed Saleh2. "Biochemical and Kinetic Study for the partially purified Lecithin: Cholesterol acyltransferase from serum cardiovascular disease." Tikrit Journal of Pure Science 25, no. 2 (March 17, 2020): 31. http://dx.doi.org/10.25130/j.v25i2.955.
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The study includes partial purification of Lecithin: Cholesterol acyltransferase (LCAT) from the blood serum of a person suffering from atherosclerosis. Several techniques were applied including ammonium sulphate precipitation, dialysis, ion exchange chromatography and electrophoresis. It was found that LCAT has one isoenzyme and the highest activity was (1073.46 × 10-3) unit/ml and a molecular weight of 62 KDa. The study also deals with characterizing of LCAT. It was found that the optimum pH and temp. of the enzyme were 7 and 35ºC.
http://dx.doi.org/10.25130/tjps.25.2020.027
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Wetsel, WC, WA Khan, I. Merchenthaler, H. Rivera, AE Halpern, HM Phung, A. Negro-Vilar, and YA Hannun. "Tissue and cellular distribution of the extended family of protein kinase C isoenzymes." Journal of Cell Biology 117, no. 1 (April 1, 1992): 121–33. http://dx.doi.org/10.1083/jcb.117.1.121.
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Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.
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Grant, Chris M., Kathryn A. Quinn, and Ian W. Dawes. "Differential Protein S-Thiolation of Glyceraldehyde-3-Phosphate Dehydrogenase Isoenzymes Influences Sensitivity to Oxidative Stress." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 2650–56. http://dx.doi.org/10.1128/mcb.19.4.2650.
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ABSTRACT The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeastSaccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.
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Przybylski, Paweł, Katarzyna Masternak, and Szymon Jastrzębowski. "Isozyme polymorphism and seed and cone variability of Scots pine (Pinus sylvestris l.) in relation to local environments in Poland." Folia Forestalia Polonica 62, no. 2 (July 1, 2020): 88–99. http://dx.doi.org/10.2478/ffp-2020-0010.
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AbstractEvolutionary processes lead to the survival of individuals best adapted to local environment. This gives rise to allele polymorphism and genetic diversity of populations. Isoenzyme proteins, which are the product of gene expression, are an effective tool for tracking these changes. On the other hand, the reproductive potential of a given population can be assessed based on its ability to produce viable and efficiently germinating seeds. The present results combine molecular analyses of isoenzyme proteins with anatomical and morphological studies of Scots pine seeds (Pinus sylvestris L.). The study was conducted in 6 populations that are characteristic of this species occurrence range in the country. The results confirm the correlation between seed weight and embryo size. They also show a population from northeastern Poland had a higher effective number of alleles and seed with lower germinative energy and capacity. There was genetic homogeneity in all except for the population from Woziwoda, which was significantly different based on the Fst test. The genetic characteristics of Scots pine from Woziwoda may be associated with the lower levels of rainfall that occur there during the growing season. The results improve our knowledge of Scots pine variability and contribute to the discussion of the impact of local environment on genetic variability.
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Parkkila, A. K., S. Parkkila, and H. Rajaniemi. "Carbonic anhydrase isoenzyme II is located in corticotrophs of the human pituitary gland." Journal of Histochemistry & Cytochemistry 44, no. 3 (March 1996): 245–50. http://dx.doi.org/10.1177/44.3.8648084.
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We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.
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Hwang, Byounghoon, Nam Ho Jeoung, and Robert A. Harris. "Pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) deficiency attenuates the long-term negative effects of a high-saturated fat diet." Biochemical Journal 423, no. 2 (September 25, 2009): 243–52. http://dx.doi.org/10.1042/bj20090390.
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The hypothesis that PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) has potential as a target for the treatment of type 2 diabetes was tested by feeding wild-type and PDHK4 knockout mice a high saturated fat diet that induces hyperglycemia, hyperinsulinaemia, glucose intolerance, hepatic steatosis and obesity. Previous studies have shown that PDHK4 deficiency lowers blood glucose by limiting the supply of three carbon gluconeogenic substrates to the liver. There is concern, however, that the increase in glucose oxidation caused by less inhibition of the pyruvate dehydrogenase complex by phosphorylation will inhibit fatty acid oxidation, promote ectopic fat accumulation and worsen insulin sensitivity. This was examined by feeding wild-type and PDHK4 knockout mice a high saturated fat diet for 8 months. Fasting blood glucose levels increased gradually in both groups but remained significantly lower in the PDHK4 knockout mice. Hyperinsulinaemia developed in both groups, but glucose tolerance was better and body weight was lower in the PDHK4 knockout mice. At termination, less fat was present in the liver and skeletal muscle of the PDHK4 knockout mice. Higher amounts of PGC-1α [PPARγ (peroxisome proliferator-activated receptor γ) coactivator 1α] and PPARα and lower amounts of fatty acid synthase and acetyl-CoA carboxylase isoenzyme 1 were present in the liver of the PDHK4 knockout mice. These findings suggest PDHK4 deficiency creates conditions that alter upstream signalling components involved in the regulation of lipid metabolism. The findings support the hypothesis that PDHK4 is a viable target for the treatment of type 2 diabetes.
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Woods, Floyd M., and Russell Pressey. "IMMUNOLOGICAL COMPARISON OF PECTINESTERASE ISOENZYMES FROM TOMATO." HortScience 25, no. 9 (September 1990): 1085e—1085. http://dx.doi.org/10.21273/hortsci.25.9.1085e.
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Pectinesterase is present in green tomato fruit and increases several-fold during ripening. Several isoenzymes of pectinesterase can be separated by chromatography of tomato extracts on DEAE-Sephadex A-50. The predominant isoenzyme in most tomato cultivars including Better Boy has been designated PE IV. This isoenzyme accounts for most of the increase in total pectinesterase during ripening of these cultivars. The fruit of some cherry tomato cultivars such as Pixie and Short Red contain some PE IV, but the major isoenzyme is PE III which occurs only in these cultivars. PE III and PE IV were isolated from ripe fruit of Short Red and Better Boy, respectively, to further characterize differences between the isoenzymes. PE III binds more strongly to cation exchangers, indicating that it is more basic than PE IV, The molecular weights were estimated by gel filtration to be 26,900 and 25, 100 for PE III and PE IV, respectively. Polyclonal antibodies were raised against the two enzymes. Cross reactivity of the enzymes with the antibodies indicates that PE III and PE IV are immunologically identical.
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Nilsen, Dennis W. T., Lasse Gøransson, Alf-Inge Larsen, Øyvind Hetland, and Peter Kierulf. "Systemic Thrombin Generation and Activity Resistant to Low Molecular Weight Heparin Administered Prior to Streptokinase in Patients with Acute Myocardial Infarction." Thrombosis and Haemostasis 77, no. 01 (1997): 057–61. http://dx.doi.org/10.1055/s-0038-1655907.
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SummaryOne hundred patients were included in a randomized open trial to assess the systemic factor Xa (FXa) and thrombin inhibitory effect as well as the safety profile of low molecular weight heparin (LMWH) given subcutaneously in conjunction with streptokinase (SK) in patients with acute myocardial infarction (MI). The treatment was initiated prior to SK, followed by repeated injections every 12 h for 7 days, using a dose of 150 anti-Xa units per kg body weight. The control group received unfractionated heparin (UFH) 12,500 IU subcutaneously every 12 h for 7 days, initiated 4 h after start of SK infusion. All patients received acetylsalicylic acid (ASA) initiated prior to SK.Serial blood samples were collected prior to and during the first 24 h after initiation of SK infusion for determination of prothrombin fragment 1+2 (Fl+2), thrombin-antithrombin III (TAT) complexes, fibrinopeptide A (FPA) and cardiac enzymes. Bleeding complications and adverse events were carefully accounted for.Infarct characteristics, as judged by creatine kinase MB isoenzyme (CK-MB) and cardiac troponin T (cTnT), were similar in both groups of patients.A comparable transient increase in Fl+2, TAT and FPA was noted irrespective of heparin regimen. Increased anti-Xa activity in patients given LMWH prior to thrombolytic treatment had no impact on indices of systemic thrombin activation.The incidence of major bleedings was significantly higher in patients receiving LMWH as compared to patients receiving UFH. However, the occurrence of bleedings was modified after reduction of the initial LMWH dose to 100 anti-Xa units per kg body weight.In conclusion, systemic FXa- and thrombin activity following SK-infusion in patients with acute MI was uninfluenced by conjunctive LMWH treatment.
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Cui, Gengyuan, Lin Li, Weixiang Xu, Mingyang Wang, Danyang Jiao, Beibei Yao, Ketao Xu, et al. "Astaxanthin Protects Ochratoxin A-Induced Oxidative Stress and Apoptosis in the Heart via the Nrf2 Pathway." Oxidative Medicine and Cellular Longevity 2020 (March 4, 2020): 1–11. http://dx.doi.org/10.1155/2020/7639109.
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This study assessed the protective mechanism of astaxanthin (ASX) against ochratoxin A- (OTA-) induced cardiac injury in mice. Four groups of mice were established: control group (0.1 mL olive oil+0.1 mL NaHCO2), OTA group (0.1 mL OTA 5 mg/kg body weight), ASX group (0.1 mL ASX 100 mg/kg body weight), and ASX + OTA group (0.1 mL ASX 100 mg/kg body weight, 2 h later, 0.1 mL OTA 5 mg/kg body weight). The test period lasted for 27 days (7 days of dosing, 2 days of rest). Electrocardiogram, body weight, heart weight, tissue pathology, oxidative markers (malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)), biochemical markers (creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH)), electron microscopy, TUNEL, and Western blot tests were used to examine the effects of OTA on myocardial injury and ASX detoxification. The results showed that OTA exposure significantly decreased both body weight and heart weight. OTA induced a decrease in heart rate in mice and decreased tissue concentrations of SOD, CAT, and GSH, while increasing serum concentrations of cardiac enzymes (CK, CK-MB, and LDH) and tissue MDA. ASX improved heart rate, cardiac enzymes, and antioxidant levels in mice. The results of tissue pathology and TUNEL assay showed that ASX protects against OTA-induced myocardial injury. In addition, Western blot results showed that the OTA group upregulated Keap1, Bax, Caspase3, and Caspase9, while it downregulated Nrf2, HO-1, and Bcl-2 protein expression. ASX played a protective role by changing the expression of Keap1, Nrf2, HO-1, Bax, Bcl-2, Caspase3, and Caspase9 proteins. These results indicate that the protective mechanism of ASX on the myocardium works through the Keap1-Nrf2 signaling pathway and mitochondria-mediated apoptosis pathway. This study provides a molecular rationale for the mechanism underlying OTA-induced myocardial injury and the protective effect of ASX on the myocardium.
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PIACENZA, L., R. RADI, F. GOÑI, and C. CARMONA. "CuZn superoxide dismutase activities from Fasciola hepatica." Parasitology 117, no. 6 (December 1998): 555–62. http://dx.doi.org/10.1017/s0031182098003394.
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The levels of superoxide dismutase (SOD) were determined in detergent-soluble, somatic and excretion–secretion (E–S) preparations from adult Fasciola hepatica using the xanthine oxidase system and visualized in substrate gels. Compared to detergent-soluble and somatic extracts, E–S products showed the highest SOD activity (88 ·5 U/mg), indicating active release to the medium in which parasites were maintained. SOD specific activity was also detected at high levels in E–S products from 3-week-old and 5-week-old immature migrating flukes (25 and 143 U/mg, respectively). In all preparations except for the somatic extract, the activity was characterized as cyanide-sensitive CuZn SOD. Differences in SOD isoenzyme profiles between the extracts were observed in native polyacrylamide gel electrophoresis: the somatic and detergent-soluble extracts exhibited 1 band of activity while the E–S products from immature and adults flukes contained 2 and 3 migrating bands, respectively. SOD was purified from the detergent-soluble extract and E–S products of adult worms by a combination of ultrafiltration, gel filtration on Sephacryl S-200 HR and ion-exchange chromatography on QAE Sephadex A-50. The SOD from detergent-soluble extract showed, by SDS–PAGE analysis, 1 band of 16 kDa apparent molecular weight. The SOD from E–S products showed 2 bands of 16 and 60 kDa apparent molecular weight. N-terminal sequence analysis of the 16 kDa band from the detergent-soluble preparation showed some similarity with Schistosoma mansoni cytoplasmic SOD. These enzymes may have a potential role in the evasion of the oxidative burst killing mechanism by immune cells.
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Music, L., and G. Sauer. "Inhibition of Moloney Murine Leukaemia Virus Transcription by a Phospholipase-C Inhibitor Affecting Trans-Acting Factors." Antiviral Chemistry and Chemotherapy 3, no. 5 (October 1992): 283–91. http://dx.doi.org/10.1177/095632029200300506.
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The propagation and the transcription of Moloney murine leukaemia virus (Mo-MuLV) can be inhibited by the antiviral compound tricyclodecan-9-yl-xanthate (code name: D609), which inhibits phospholipase-C (PLC) and, as a consequence, the activation of protein kinase-C (PKC) isoenzyme(s). The frans-acting factors LVa, LVb, and LVc were shown to be affected; it was not possible to retrieve them after treatment with D609 from Mo-MuLV producer cells, by virtue of the binding affinity to their consensus sequences. In contrast, the binding efficiency of the other three known transacting factors (core, NF1 and GRE), which in addition to the viral transcription, play a role in the regulation of cellular mRNA synthesis remained unimpaired. Neither LVa, LVb, nor LVc was found to be phosphorylated, which suggests that these are not targets of PKC. Only one phosphorylated DNA-binding protein was identified with an apparent molecular weight of 34kDa. This protein co-purified irrespective of the recognition sequences that we used (LVa, LVb, LVc, core, and NF1). Direct evidence is provided for the inhibition of the TPA-induced phosphorylation of the 34 kDa protein by D609. We suggest that the binding of LVa, LVb, and LVc to the DNA is mediated by the 34 kDa protein in its phosphorylated state.
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Thomas, M. R., J. P. Miell, A. M. Taylor, R. J. M. Ross, J. R. Arnao, D. E. Jewitt, and A. M. McGregor. "Endocrine and cardiac paracrine actions of insulin-like growth factor-I (IGF-I) during thyroid dysfunction in the rat: is IGF-I implicated in the mechanism of heart weight/body weight change during abnormal thyroid function?" Journal of Molecular Endocrinology 10, no. 3 (June 1993): 313–23. http://dx.doi.org/10.1677/jme.0.0100313.
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ABSTRACT Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function. Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10−3, P<0·01) and treated hypothyroid (2·87±0·07 ×10−3, P<0·001) animals when compared with controls (2·26±0·03 × 10−3). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg). Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.
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Rovner, A. S., M. M. Thompson, and R. A. Murphy. "Two different heavy chains are found in smooth muscle myosin." American Journal of Physiology-Cell Physiology 250, no. 6 (June 1, 1986): C861—C870. http://dx.doi.org/10.1152/ajpcell.1986.250.6.c861.
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Two putative myosin heavy chains designated SM1 and SM2 were detected on a 3.5% polyacrylamide-sodium dodecyl sulfate gel electrophoresis system loaded with homogenates of several mammalian smooth muscles. The two polypeptides were present in nearly equal amounts in all smooth muscle tissues tested and in myosin purified from swine carotid media and stomach. Both proteins were equally stained by smooth muscle-specific myosin antibodies. The smaller of the polypeptides had a mobility nearly identical to that of the single heavy chain observed in purified fast-twitch skeletal myosin. Electrophoresis of pyrophosphate extracts from swine carotid media, swine stomach, rabbit thoracic aorta, and guinea pig taenia coli on nondenaturing pyrophosphate gels revealed a single protein band. When subsequently electrophoresed on a sodium dodecyl sulfate gel, the native bands from swine tissue extracts revealed the two putative heavy chains in nearly equal amounts, as well as a large amount of a higher molecular weight peptide whose properties reflect those of filamen. Sodium dodecyl sulfate gel analysis of the myosin band from pyrophosphate gels of purified swine stomach myosin showed exclusively the two heavy chains in a nearly 1:1 ratio. Smooth muscle myosin migrates homogeneously on pyrophosphate gels, and the virtual equality of the two heavy chains may reflect the presence of large amounts of a myosin isoenzyme, which is a heavy-chain heterodimer.
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Quayle, Julie A., Alison Capstick, Anthony I. Morris, and David Billington. "Plasma alkaline phosphodiesterase I in intrahepatic cholestasis induced by α-naphthylisothiocyanate in rats." Clinical Science 75, no. 1 (July 1, 1988): 13–20. http://dx.doi.org/10.1042/cs0750013.
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1. Administration of α-naphthylisothiocyanate (ANIT) to rats produced dose-dependent increases in plasma bile acid and bilirubin concentrations. Similar increases in plasma bile acid and bilirubin concentrations were evident in bile duct ligated rats, indicating that the severity of cholestasis is almost identical in both models. 2. Plasma alkaline phosphodiesterase I was increased by only 50–80% while alkaline phosphatase was increased more than threefold after ANIT administration. This is in contrast to an earlier study [S. R. Simpson, K. Rahman & D. Billington (1984) Clinical Science 67, 647–652] where, after bile duct ligation, serum alkaline phosphodiesterase I was elevated sixfold before any increase in alkaline phosphatase activity became apparent. Thus, plasma alkaline phosphodiesterase I does not offer as sensitive a marker of intrahepatic cholestasis (induced by ANIT) as it does of extrahepatic cholestasis (induced by bile duct ligation). 3. Hepatic alkaline phosphodiesterase I was unaffected by ANIT pretreatment while hepatic alkaline phosphatase was increased up to seven times. It is suggested that raised plasma alkaline phosphodiesterase I is due to regurgitation of the biliary enzyme rather than overspill of the enzyme from liver into blood. 4. Gel filtration showed that 24 h and 96 h after ANIT administration, rat serum contained a high molecular weight form of alkaline phosphodiesterase I, suggesting a different isoenzyme profile.
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Bin Jardan, Yousef A., Mushtaq Ahmad Ansari, Mohammad Raish, Khalid M. Alkharfy, Abdul Ahad, Fahad I. Al-Jenoobi, Nazrul Haq, Mohd Rashid Khan, and Ajaz Ahmad. "Sinapic Acid Ameliorates Oxidative Stress, Inflammation, and Apoptosis in Acute Doxorubicin-Induced Cardiotoxicity via the NF-κB-Mediated Pathway." BioMed Research International 2020 (March 10, 2020): 1–10. http://dx.doi.org/10.1155/2020/3921796.
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In the present study, we explored SA’s activity against DOX-induced cardiotoxicity and revealed its underlying mechanisms. Male Wistar rats (weight, 190-210g; n=6) were randomly divided into four groups: group I, normal control; group II, DOX 15 mg/kg via intraperitoneal (ip) route; group III, administered DOX+SA 20 mg/kg; and group IV, administered DOX+captopril (CAP 30 mg/kg). SA and CAP were administered orally for seven days, and DOX (15 mg/kg) was injected intraperitoneally an hour before SA treatment on the fifth day. Forty-eight hours after DOX administration, animals were anesthetized and sacrificed for molecular and histology experiments. SA significantly mitigated the myocardial effects of DOX, and following daily administration, it reduced serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme-MB to near normal values. Levels of oxidative stress markers, glutathione-peroxidase, superoxide dismutase, and catalase, in the cardiac tissue were significantly increased, whereas malondialdehyde levels decreased after SA treatment in DOX-administered rats. Furthermore, DOX caused an inflammatory reaction by elevating the levels of proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and endothelin- (ET-) 1, as well as nuclear factor kappa-B (NF-κB) expression. Daily administration of SA significantly repressed TNF-α, IL-1β, ET-1, and NF-κB levels. caspase-3 and Bax expression, bcl-2-like protein and caspase-3 activities and levels. Overall, we found that SA could inhibit DOX-induced cardiotoxicity by inhibiting oxidative stress, inflammation, and apoptotic damage.
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Barceló, Alfonso Ros, Federico Pomar, Matías López-Serrano, and Maria Angeles Pedreño. "Peroxidase: a multifunctional enzyme in grapevines." Functional Plant Biology 30, no. 6 (2003): 577. http://dx.doi.org/10.1071/fp02096.
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Peroxidases are heme-containing enzymes that catalyse the one-electron oxidation of several substrates at the expense of H2O2. They are probably encoded by a large multigene family in grapevines, and therefore show a high degree of polymorphism. Grapevine peroxidases are glycoproteins of high thermal stability, whose molecular weight usually ranges from 35 to 45 kDa. Their visible spectrum shows absorption bands characteristic of high-spin class III peroxidases. Grapevine peroxidases are capable of accepting a wide range of natural compounds as substrates, such as the cell wall protein extensin, plant growth regulators such as IAA, and phenolics such as benzoic acids, stilbenes, flavonols, cinnamyl alcohols and anthocyanins. They are located in cell walls and vacuoles. These locations are in accordance with their key role in determining the final cell wall architecture, especially regarding lignin deposition and extensin insolubilization, and the turnover of vacuolar phenolic metabolites, a task that also forms part of the molecular program of disease resistance. Although peroxidase is a constitutive enzyme in grapevines, its levels are strongly modulated during plant cell development and in response to both biotic and abiotic environmental factors. To gain an insight into the metabolic regulation of peroxidase, several authors have studied how grapevine peroxidase and H2O2 levels change in response to a changing environment. Nevertheless, the results obtained are not always easy to interpret. Despite such difficulties, the response of the peroxidase–H2O2 system to both UV-C radiation and Trichoderma viride elicitors is worthy of study. Both UV-C and T. viride elicitors induce specific changes in peroxidase isoenzyme / H2O2 levels, which result in specific changes in grapevine physiology and metabolism. In the case of T. viride-elicited grapevine cells, they show a particular mechanism for H2O2 production, in which NADPH oxidase-like activities are apparently not involved. However, they offer a unique system whereby the metabolic regulation of peroxidase by H2O2, with all its cross-talks and downstream signals, may be elegantly dissected.
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Łopieńska-Biernat, E., K. Żółtowska, and J. Rokicki. "Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages ofAnisakis simplex." Journal of Helminthology 82, no. 1 (March 2008): 45–51. http://dx.doi.org/10.1017/s0022149x0787355x.
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AbstractExtracts ofAnisakis simplexthird (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of α-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS–PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: α1, α2 and α3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The α-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations ofA. simplexlarvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.
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Archid, Solass, Tempfer, Königsrainer, Adolph, Reymond, and Wilson. "Cachexia Anorexia Syndrome and Associated Metabolic Dysfunction in Peritoneal Metastasis." International Journal of Molecular Sciences 20, no. 21 (October 31, 2019): 5444. http://dx.doi.org/10.3390/ijms20215444.
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: Patients with peritoneal metastasis (PM) of gastrointestinal and gynecological origin present with a nutritional deficit characterized by increased resting energy expenditure (REE), loss of muscle mass, and protein catabolism. Progression of peritoneal metastasis, as with other advanced malignancies, is associated with cancer cachexia anorexia syndrome (CAS), involving poor appetite (anorexia), involuntary weight loss, and chronic inflammation. Eventual causes of mortality include dysfunctional metabolism and energy store exhaustion. Etiology of CAS in PM patients is multifactorial including tumor growth, host response, cytokine release, systemic inflammation, proteolysis, lipolysis, malignant small bowel obstruction, ascites, and gastrointestinal side effects of drug therapy (chemotherapy, opioids). Metabolic changes of CAS in PM relate more to a systemic inflammatory response than an adaptation to starvation. Metabolic reprogramming is required for cancer cells shed into the peritoneal cavity to resist anoikis (i.e., programmed cell death). Profound changes in hexokinase metabolism are needed to compensate ineffective oxidative phosphorylation in mitochondria. During the development of PM, hypoxia inducible factor-1α (HIF-1α) plays a key role in activating both aerobic and anaerobic glycolysis, increasing the uptake of glucose, lipid, and glutamine into cancer cells. HIF-1α upregulates hexokinase II, phosphoglycerate kinase 1 (PGK1), pyruvate dehydrogenase kinase (PDK), pyruvate kinase muscle isoenzyme 2 (PKM2), lactate dehydrogenase (LDH) and glucose transporters (GLUT) and promotes cytoplasmic glycolysis. HIF-1α also stimulates the utilization of glutamine and fatty acids as alternative energy substrates. Cancer cells in the peritoneal cavity interact with cancer-associated fibroblasts and adipocytes to meet metabolic demands and incorporate autophagy products for growth. Therapy of CAS in PM is challenging. Optimal nutritional intake alone including total parenteral nutrition is unable to reverse CAS. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) stabilized nutritional status in a significant proportion of PM patients. Agents targeting the mechanisms of CAS are under development.
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Mateus, Rogério Pincela, Hamilton Cabral, Gustavo Orlando Bonilla-Rodriguez, and Carlos Roberto Ceron. "Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis." Brazilian Archives of Biology and Technology 52, no. 5 (October 2009): 1083–89. http://dx.doi.org/10.1590/s1516-89132009000500004.
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A method that allows the measure of molecular weight of two well-known and closely related esterases from Drosophila mojavensis and its sibling species, D. arizonae, is here described, using native polyacrylamide gel electrophoresis at several concentrations, applying Fergunson´s principles. These enzymes, namely EST-4 and EST-5, presented molecular weight values between 81 and 91 kDa. In spite of their distinct expression pattern through the insect's life cycle, they showed properties of isoenzymes codified by distinct structural genes, supporting the hypothesis of a rather recent gene duplication event that generated both in D. mojavensis and D. arizonae, as well as in other species of repleta group. The method is simple and adequate to be applied to preliminary molecular weight determination of other enzymes without any previous purification procedure.
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Macias, Pedro, and M. Carmen Pinto. "Purification and Partial Characterization of Rat Liver Lipoxygenase." Zeitschrift für Naturforschung B 42, no. 10 (October 1, 1987): 1343–48. http://dx.doi.org/10.1515/znb-1987-1020.
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Abstract Lipoxygenase was purified from rat liver cytosolic fraction by a method involving two successive chromatographic steps on Sephacryl S-200 and Phenyl Sepharose CL-4B. The enzyme has a molecular weight of 96 Kdal and it seems to be composed of two identical subunits. Chromatofocusing of the enzyme revealed a single band of activity at pi 6.3. The enzyme activity of the purified fraction showed maximum activity at pH 7.0 with a Km for linoleic acid of 1.4 μM and is competitively inhibited by the specific lipoxygenase inhibitor nordihydroguaiaretic acid. The purified enzyme shows absorption and fluorescence spectra similar to those of lipoxygenase from other sources. However, the molecular weight of lipoxygenase purified from liver is found to be different from that of the enzyme from polymorphonuclear leukocytes. It is suggested that there are different isoenzymes of lipoxygenases in mammals.
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Buat, M. L., G. Landemore, and J. Izard. "Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study." Journal of Histochemistry & Cytochemistry 36, no. 9 (September 1988): 1109–15. http://dx.doi.org/10.1177/36.9.3403966.
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We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.
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Park, Hee Woo, Ki Seok Koh, and Soon Cheol Park. "Molecular weights and inhibitor sensitivities of alkaline phosphatase isoenzymes from the midgut of the earthworm, Eisenia andrei." Soil Biology and Biochemistry 30, no. 6 (June 1998): 831–32. http://dx.doi.org/10.1016/s0038-0717(97)00160-0.
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Ryan, Frederick J., and Debra R. Ayres. "Molecular markers indicate two cryptic, genetically divergent populations of Russian thistle (Salsola tragus) in California." Canadian Journal of Botany 78, no. 1 (March 7, 2000): 59–67. http://dx.doi.org/10.1139/b99-160.
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Genetic variability among accessions of Russian thistle (Salsola tragus L.) from California was investigated using allozymes and DNA-based molecular markers. Aspartate aminotransferase and 6-phosphogluconate dehydrogenase displayed two multienzyme phenotypes that were widespread in plants throughout the state. Random amplified polymorphic DNA analysis was conducted on samples of the two isoenzymic phenotypes collected throughout California, as well as additional accessions from France and Turkey and Salsola paulsenii Litv. Six primers produced 23 polymorphic bands. Analysis of the patterns of bands by calculation of simple matching coefficients and cluster analysis confirmed the genetic distinctness of the two isoenzymic phenotypes of S. tragus; S. paulsenii was markedly different from both types. Mean fruit weights from plants grown under similar conditions were different between the two types as well. These results and preliminary cytological analysis together suggest that the two types are actually two different species of Salsola, only one of which has been previously recognized. Analysis of the DNA-based markers suggests that one of the genetic entities may be closely related to Salsola found in Europe, while the area of origin of the second entity is currently obscure.Key words: allozyme, genetic diversity, RAPD assay, Salsola tragus, Salsola paulsenii.
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Cornford, C. A., and R. D. Hill. "α-Amylase isoenzymes and BASI-like proteins in seeds of different grass species." Seed Science Research 4, no. 3 (September 1994): 285–91. http://dx.doi.org/10.1017/s0960258500002312.
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AbstractSeeds from an assortment of 28 grass species (including two cereals), chiefly representing different tribes of the sub-family Pooideae were tested for the presence of BASI-like proteins using Western blotting techniques and antisera raised against the barley amylase/subtilisin inhibitor. A single protein species of the same molecular weight as the BASI protein was detected in each member of the tribes Triticeae and Bromeae tested. Members of the Triticeae and Bromeae were the only species to produce high pi α-amylase isoenzymes following germination of the grain in addition to low pl forms which were present in all species tested. Anti-BASI antibody binding patterns for the other grasses examined were variable with the strongest staining being observed for Lolium species. In these species, either a double or single protein band fractionally larger than the BASI protein was recognized. In most other cases antibody binding was barely or not detectable. Inhibitor preparations from Hordeum vulgare and Lolium perenne were effective at inhibiting wheat α-amylase but neither had any effect against enzyme produced by germinating L. perenne.
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Terkeltaub, Robert A. "Inorganic pyrophosphate generation and disposition in pathophysiology." American Journal of Physiology-Cell Physiology 281, no. 1 (July 1, 2001): C1—C11. http://dx.doi.org/10.1152/ajpcell.2001.281.1.c1.
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Inorganic pyrophosphate (PPi) regulates certain intracellular functions and extracellular crystal deposition. PPiis produced, degraded, and transported by specialized mechanisms. Moreover, dysregulated cellular PPiproduction, degradation, and transport all have been associated with disease, and PPiappears to directly mediate specific disease manifestations. In addition, natural and synthetic analogs of PPiare in use or currently under evaluation as prophylactic agents or therapies for disease. This review summarizes recent developments in the understanding of how PPiis made and disposed of by cells and assesses the body of evidence for potentially significant physiological functions of intracellular PPiin higher organisms. Major topics addressed are recent lines of molecular evidence that directly link decreased and increased extracellular PPilevels with diseases in which connective tissue matrix calcification is disordered. To illustrate in depth the effects of disordered PPimetabolism, this review weighs the roles in matrix calcification of the transmembrane protein ANK, which regulates intracellular to extracellular movement of PPi, and the PPi-generating phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1).
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Mirhashemi, S. M., Ali-Asghar Moshtaghie, M. Ani, and Mohammad Hossein Aa. "Lead Toxicity on Kinetic Behaviors of High and Low Molecular Weight Alkaline Phosphatase Isoenzymes of Rat, in vivo and in vitro Studies." Journal of Biological Sciences 10, no. 4 (May 1, 2010): 341–47. http://dx.doi.org/10.3923/jbs.2010.341.347.
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Oesch, F., J. Doehmer, T. Friedberg, H. R. Glatt, B. Oesch-Bartlomowicz, K. L. Platt, P. Steinberg, D. Utesch, and H. Thomas. "Toxicological Implications of Enzymatic Control of Reactive Metabolites." Human & Experimental Toxicology 9, no. 3 (May 1990): 171–77. http://dx.doi.org/10.1177/096032719000900309.
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Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.
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Zhao, Mingcai, Cindy Sutherland, David P. Wilson, Jingti Deng, Justin A. MacDonald, and Michael P. Walsh. "Identification of the linker histone H1 as a protein kinase Cε-binding protein in vascular smooth muscle." Biochemistry and Cell Biology 82, no. 5 (October 1, 2004): 538–46. http://dx.doi.org/10.1139/o04-053.
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A variety of anchoring proteins target specific protein kinase C (PKC) isoenzymes to particular subcellular locations or multimeric signaling complexes, thereby achieving a high degree of substrate specificity by localizing the kinase in proximity to specific substrates. PKCε is widely expressed in smooth muscle tissues, but little is known about its targeting and substrate specificity. We have used a Far-Western (overlay) approach to identify PKCε-binding proteins in vascular smooth muscle of the rat aorta. Proteins of ~32 and 34 kDa in the Triton-insoluble fraction were found to bind PKCε in a phospholipid/diacylglycerol-dependent manner. Although of similar molecular weight to RACK-1, a known PKCε-binding protein, these proteins were separated from RACK-1 by SDS-PAGE and differential NaCl extraction and were not recognized by an antibody to RACK-1. The PKCε-binding proteins were further purified from the Triton-insoluble fraction and identified by de novo sequencing of selected tryptic peptides by tandem mass spectrometry as variants of the linker histone H1. Their identity was confirmed by Western blotting with anti-histone H1 and the demonstration that purified histone H1 binds PKCε in the presence of phospholipid and diacylglycerol but absence of Ca2+. The interaction of PKCε with histone H1 was specific since no interaction was observed with histones H2A, H2S or H3S. Bound PKCε phosphorylated histone H1 in a phospholipid/diacylglycerol-dependent but Ca2+-independent manner. Ca2+-dependent PKC was also shown to interact with histone H1 but not other histones. These results suggest that histone H1 is both an anchoring protein and a substrate for activated PKCε and other PKC isoenzymes and likely serves to localize activated PKCs that translocate to the nucleus in the vicinity of specific nuclear substrates including histone H1 itself. Since PKC isoenzymes have been implicated in regulation of gene expression, stable interaction with histone H1 may be an important step in this process.Key words: protein kinase C, histone H1, signaling complexes, smooth muscle.
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Nam, Trinh Ngoc, Nguyen Nhat Vinh, Le Hong Thia, and Tran Do Kim Hue. "Transcriptome analysis of copper stress response in rice seedling using DNA microarray." Vietnam Journal of Biotechnology 14, no. 4 (April 19, 2018): 629–44. http://dx.doi.org/10.15625/1811-4989/14/4/12296.
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Heavy metal contamination along with the increase in food demand are a primary concern in Vietnam and all over the world. In order to enhance crop tolerance to unfavorable cultivation conditions including heavy metal toxicity, understanding of plant response system under the effect of heavy metals is necessary. In the current study, physiological, biochemical and transcriptomic changes of rice seedings (Oryza sativa L. cv. IR64) were investigated under copper (Cu) stress. Root elongation and root fresh weight were decreased whereas accumulation of copper in root was enhanced significantly with increasing copper concentration from 2.5 to 15 M. In addition, copper induced endogenous reactive oxygen species (ROS) generation and activated isoenzymes of superoxide dismutase (SOD) and catalase (CAT). The molecular mechanism of rice roots in response to copper toxicity at mRNA expression level was analyzed by microarray technique. Functions and roles of genes were also analyzed by bioinformatic tools AgriGO and MapMan. Gene ontology analysis revealed that 1900 Cu responsive genes were involved in phytohormones, reactive oxygen species, signaling pathways, transcription factors, transport activities, antioxidant defense systems. Through phytohormones and reactive oxygen species, Cu may inhibit rice root growth. Phytohormones and reactive oxygen species can also be signal molecules in signaling pathways with the participation of mitogen-activated protein kinase (MAPK) cascades, and transcription factors in response to Cu stress. Detoxification and protection mechanisms may involve transport activities and antioxidant defense systems during Cu treatment. These results may provide new insights into mechanisms of rice plant to tolerate with Cu toxicity conditions.
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Hulea, Stefan A., Henry R. V. Arnstein, and Fred A. Kummerow. "Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis." Analytical Letters 27, no. 9 (July 1994): 1703–11. http://dx.doi.org/10.1080/00032719408007428.
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Ahmad, Areeba, Mohd Irshad, Waseem Ahmad, Abdul Khan, and Riaz Ahmad. "Analysis of Serum Proteins and Enzymes Level in Human Subjects with Osteoarthritis." Journal of Medical Biochemistry 30, no. 1 (January 1, 2011): 15–24. http://dx.doi.org/10.2478/v10011-010-0045-4.
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Analysis of Serum Proteins and Enzymes Level in Human Subjects with OsteoarthritisThe aim of the present study was to assess the serum proteins and enzymes level using polyacrylamide gel electrophoretic (PAGE) profiles in human subjects with osteoarthritis (OA). Forty-one subjects with confirmed OA were selected for the present study. Sera were collected from these individuals and loaded in equal amounts on native and denaturing PAGE separately. Software analysis of these profiles was done using Scion Imaging (Beta release-4, Scion Corporation) and GelPro (Media Cybernetics, USA) programs. To visualize esterases (Est) and lactate dehydrogenase (LDH) isoenzymes in the sera of these patients substrate specific staining was performed. Differences in the values of control and OA subjects were tested statistically. Software analysis of native-PAGE profiles revealed the presence of nineteen peptides in control and twenty one in OA subjects respectively. Two extra peptides were present in the β-globulins region of OA subjects. Significant decline from 42.77% to 34.72% in albumin levels (hypoalbuminemia) was observed in OA subjects with total albumin to globulin ratio 0.58. In SDS-PAGE, the difference in control and OA subjects was observed among eight peptides with molecular weight 25, 22 and 20 kDa (absent in OA) and five novel peptides 270, 125, 30, 21.36 and 18.4 kDa (absent in controls), while albumin retains the major activity. For enzymes, Est follow a relative order, BchEst (42.86%)> ArylEst (16.24%)>AchEst (6.85%) in OA subjects with the expression of a new BchEst isoform in 4.78% and two isoforms of ArylEst at 2.13 and 1.61% concentrations respectively. Significantly declined albumin esterase-like activity (AlbEst) was observed (34%) (P<0.05) in diseased subjects compared with controls (47%). Significant increase in LDH-5 and decline in LDH-1 and -2 isoenzymes were also observed in the sera of OA subjects. However, the overall rank of LDH isoenzymes was similar in control and OA subjects. Our results demonstrate noticeable differences in the sera PAGE profiles and enzymes activity in control and OA subjects and provide evidence to select serum for its use in the search for suitable biochemical markers in osteoarthritis.
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Ikemoto, Masaki, Masayoshi Tabata, Takashi Murachi, and Masayuki Totani. "Purification and Properties of Human Erythrocyte Arginase." Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 26, no. 6 (November 1989): 547–53. http://dx.doi.org/10.1177/000456328902600616.
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An efficient method for purification of human erythrocyte arginase was developed. This method included two new procedures, hydrophobic chromatography and immunoaffinity chromatography, and yielded 0·7 mg of homogeneous arginase protein from 2·1 L of haemolysate. The molecular weight of native arginase was estimated to be 105 000 by gel filtration on a Sephadex G-150 column, and that of its subunit 35 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This indicates that the native enzyme is composed of three homologous subunits. Amino acid composition of human erythrocyte arginase was found to be very similar to that of liver arginase of several other mammals. After dialysis against distilled water, the purified arginase still retained its enzymatic activity which was decreased by EDTA and reversibly restored by Mn(II) ion. A specific polyclonal antibody for use in an immunoassay was also produced. This antibody revealed one single band on immunoelectrophoretic analysis of the acetone powder extract, suggesting absence of arginase isoenzymes in human erythrocytes.
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Ward, Robert D., and David O. F. Skibinski. "Evidence that mitochondrial isozymes are genetically less variable than cytoplasmic isozymes." Genetical Research 51, no. 2 (April 1988): 121–27. http://dx.doi.org/10.1017/s0016672300024137.
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SummaryIt has been proposed that isoenzymes functioning within cell organelles (chloroplasts, mitochondria) are genetically less variable than their cytoplasmic counterparts, as a result either of constraints imposed by the need to cross organelle membranes or from the different and specialized nature of organelle metabolism. However, some recent findings concerning chloroplast and cytoplasmic isozyme variability are not consistent with this thesis. We have analyzed a number of surveys of electrophoretically detectable enzyme variation in vertebrates, and show that for each of the four tested enzymes (malate dehydrogenase, isocitrate dehydrogenase, malic enzyme, and aspartate aminotransferase), the mitochondrial isozymes are less variable than their corresponding cytosplasmic forms. The mean heterozygosities across the four enzymes are 0·083 and 0·038 for the cytoplasmic and mitochondrial forms respectively. We conclude that mitochondrial isozymes are indeed subject to greater constraints than cytoplasmic forms and have fewer sites able to accept neutral or slightly deleterious mutations. It is also noted that of the enzymes analyzed, that with the smallest subunit molecular weight (MDH) has the least variable cytoplasmic and mitochondrial isozymes, whereas the enzyme with the largest subunits (ME) has the most variable isozymes.
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Ma, Zhongnv, Xianbao Shi, Gang Zhang, Feng Guo, Lina Shan, and Jiqun Cai. "Metabolism and Metabolic Inhibition of Xanthotoxol in Human Liver Microsomes." Evidence-Based Complementary and Alternative Medicine 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/5416509.
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Cytochrome p450 (CYP450) enzymes are predominantly involved in Phase I metabolism of xenobiotics. In this study, the CYP450 isoforms involved in xanthotoxol metabolism were identified using recombinant CYP450s. In addition, the inhibitory effects of xanthotoxol on eight CYP450 isoforms and its pharmacokinetic parameters were determined using human liver microsomes. CYP1A2, one of CYP450s, played a key role in the metabolism of xanthotoxol compared to other CYP450s. Xanthotoxol showed stronger inhibition on CYP3A4 and CYP1A2 compared to other isoenzymes with the IC50of 7.43 μM for CYP3A4 and 27.82 μM for CYP1A2. The values of inhibition kinetic parameters (Ki) were 21.15 μM and 2.22 μM for CYP1A2 and CYP3A4, respectively. The metabolism of xanthotoxol obeyed the typical monophasic Michaelis-Menten kinetics andVmax,Km, andCLintvalues were calculated as 0.55 nmol·min−1·mg−1, 8.46 μM, and 0.06 mL·min−1·mg−1. In addition, the results of molecular docking showed that xanthotoxol was bound to CYP1A2 with hydrophobic andπ-πbond and CYP3A4 with hydrogen and hydrophobic bond. We predicted the hepatic clearance (CLH) and theCLHvalue was 15.91 mL·min−1·kg−1body weight. These data were significant for the application of xanthotoxol and xanthotoxol-containing herbs.
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Souza, Isabel Regina Prazeres de, Vera Maria Carvalho Alves, Sidney Netto Parentoni, Antônio Carlos de Oliveira, Flávia França Teixeira, Jennifer Wilson MacAdam, and Antônio Álvaro Corsetii Purcino. "Change in root apical protein and peroxidase activity in response to aluminum in tolerant and sensitive maize inbred lines." Brazilian Journal of Plant Physiology 14, no. 3 (September 2002): 219–24. http://dx.doi.org/10.1590/s1677-04202002000300006.
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The effects of a short-term (80 min) exposure to 222 µM aluminum (Al) on the protein content and expression and on peroxidase activity and isoenzymes in the primary root of maize were evaluated. Two inbred lines differing in their level of tolerance to Al were used: Cateto 237 (tolerant) and L36 (sensitive). The apical 20 mm of the primary root was divided into 2-mm-long segments that were analyzed for total protein content and peroxidase activity. These results demonstrate that the total protein content along the root apex was not affected by Al in the tolerant inbred line, but decreased in the sensitive line. In the apical 2 mm of the root of the sensitive line, the expression of low molecular weight proteins (43 kDa or smaller) was decreased. Expression of low molecular proteins increased in the tolerant inbred line, even though total protein content did not increase. This suggests that some of these proteins could play a role in metal tolerance, perhaps as binding peptides. While the peroxidase activity of the tolerant inbred line did not change with exposure to Al, peroxidase activity in the apical 6 mm of the root of the sensitive line decreased. The tolerant inbred line constitutively expressed more anionic peroxidase isoforms. These results demonstrate that maintenance of protein expression may be an important component of the plant's resistance to Al stress, and that resistance to Al stress is associated with the higher expression of anionic peroxidase isoforms.
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Wu, Weisheng, Jie Lu, Yamin Wei, Jin Wang, Juan Lin, Shuwen Cao, Xiaofen Sun, and Kexuan Tang. "Isolation and Characterization of the First Putative Peroxidase Gene from Oilseed Rape (Brassica napus) which is Highly Homologous to HRPC." Bioscience Reports 26, no. 3 (August 18, 2006): 263–80. http://dx.doi.org/10.1007/s10540-006-9021-0.
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A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca2+ sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H2O2. The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC.
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harIkya, Julius, Charles Charles, and James Ayatse. "Characterization of Genetically Engineered Linamarase (β-glucosidase) from Saccharomyces cerevisiae." Current Research in Nutrition and Food Science Journal 1, no. 2 (November 26, 2013): 139–45. http://dx.doi.org/10.12944/crnfsj.1.2.05.
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The characterization parameters of genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae due to action of the enzyme on linamarin as influenced by degree of purification, pH and temperature were investigated. Commercial native linamarase (CNLIN) was used as control. Linamarase genes (chromosomal DNA) and plasmids (circular DNA) isolated from bitter cassava and yeast respectively were restricted and ligated to produce recombinant genes (r-DNA). The r-DNA were introduced into the nucleus of CaCl2 induced competent Saccharomyces cerevisiae cells which transformed into strains capable of producing genetically engineered linamarase (GELIN). Recombinant S. cerevisiae cells at the stationary phase of growth were recovered, homogenized and centrifuged to obtain crude extracts designated as GELIN0. Carboxy methyl cellulose, diethyl amino-ethyl-sephadex and diethyl amino-ethyl-cellulose were used to purify the crude extracts resulting in GELIN1, GELIN2 and GELIN3, respectively. The physical characterization parameters of the enzyme extracts such as impurity levels, molecular weights (Mwt), number of isoenzyme, sulphur amino acids (methionine and cysteine) and the electrical charges were evaluated using standard methods. The ability of the enzyme extracts and a commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged using linamarin (cassava) as substrates for characterization of activity kinetic profiles such as optimum pH (pHopt), temperature (Topt), total activity, specific activity, purity fold, yield and efficiency ratio. The results indicated that the genetically engineered linamarase(β-glucosidase) consisted of 3 isoenzyme forms. Purification conferred different ionic charges of zero to GELIN0, unit positive charge GELIN1, and unit negative charge to GELIN2 and GELIN3 respectively. Ranges for other parameters were Mwt (22,000-26,000 Daltons), insoluble protein impurity (0.4 -3.5 mg/100g sample) and purity fold (11.5 -1.0) for GELIN3 - GELIN0). Methionine and cystiene varied from 2.0 to 2.6% and 3.0 to 20% respectively (CNLIN - GELIN3). The native commercial enzyme (CNLIN) acted only at pH 6.8 on linamarin with pHopt and Topt of 6.8 and 35 oC respectively. The wide pH tolerance and specific activity towards linamarin degradation suggest a possible use of the genetically engineered linamarase from S. cerevisiae in detoxification of cassava for increased production exportation of cassava-based food products.
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Hung, Ming-Ni, Zhicheng Xia, Nien-Tai Hu, and Byong H. Lee. "Molecular and Biochemical Analysis of Two β-Galactosidases from Bifidobacterium infantisHL96." Applied and Environmental Microbiology 67, no. 9 (September 1, 2001): 4256–63. http://dx.doi.org/10.1128/aem.67.9.4256-4263.2001.
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ABSTRACT Two genes encoding β-galactosidase isoenzymes,β-galI and β-galIII, fromBifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. β-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5′ flanking region of β-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently fromβ-galI. The β-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of β-GalI and β-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in β-GalI, and a possible acid-base site proposed for the LacG family was located in β-GalIII, which featured a glutamate at residue 160. The coding regions of the β-galI andβ-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. β-GalI and β-GalIII were specific for β-d-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-β-d-galactopyranoside) than in response to lactose, particularly β-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by β-GalI at 37°C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with β-GalIII. The structure of the major trisaccharide produced by β-GalI catalysis was characterized asO-β-d-galactopyranosyl-(1-3)-O-β-d-galactopyranosyl-(1-4)-d-glucopyranose (3′-galactosyl-lactose).
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Elblehi, Samar S., Yasser S. El-Sayed, Mohamed Mohamed Soliman, and Mustafa Shukry. "Date Palm Pollen Extract Avert Doxorubicin-Induced Cardiomyopathy Fibrosis and Associated Oxidative/Nitrosative Stress, Inflammatory Cascade, and Apoptosis-Targeting Bax/Bcl-2 and Caspase-3 Signaling Pathways." Animals 11, no. 3 (March 20, 2021): 886. http://dx.doi.org/10.3390/ani11030886.
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Doxorubicin (DOX) has a potent antineoplastic efficacy and is considered a cornerstone of chemotherapy. However, it causes several dose-dependent cardiotoxic results, which has substantially restricted its clinical application. This study was intended to explore the potential ameliorative effect of date palm pollen ethanolic extract (DPPE) against DOX-induced cardiotoxicity and the mechanisms underlying it. Forty male Wistar albino rats were equally allocated into Control (CTR), DPPE (500 mg/kg bw for 4 weeks), DOX (2.5 mg/kg bw, intraperitoneally six times over 2 weeks), and DPPE + DOX-treated groups. Pre-coadministration of DPPE with DOX partially ameliorated DOX-induced cardiotoxicity as DPPE improved DOX-induced body and heart weight changes and mitigated the elevated cardiac injury markers activities of serum aminotransferases, lactate dehydrogenase, creatine kinase, and creatine kinase-cardiac type isoenzyme. Additionally, the concentration of serum cardiac troponin I (cTnI), troponin T (cTnT), N-terminal pro-brain natriuretic peptide (NT-pro BNP), and cytosolic calcium (Ca+2) were amplified. DPPE also alleviated nitrosative status (nitric oxide) in DOX-treated animals, lipid peroxidation and antioxidant molecules as glutathione content, and glutathione peroxidase, catalase, and superoxide dismutase activities and inflammatory markers levels; NF-κB p65, TNF-α, IL-1β, and IL-6. As well, it ameliorated the severity of histopathological lesions, histomorphometric alteration and improved the immune-staining of the pro-fibrotic (TGF-β1), pro-apoptotic (caspase-3 and Bax), and anti-apoptotic (Bcl-2) proteins in cardiac tissues. Collectively, pre-coadministration of DPPE partially mitigated DOX-induced cardiac injuries via its antioxidant, anti-inflammatory, anti-fibrotic, and anti-apoptotic potential.
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Reiprich, Sebastian, Eva Hofbauer, Stefanie Kiderlen, Hauke Clausen-Schaumann, Wolfgang Böcker, Attila Aszódi, and Veronika Schönitzer. "Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells." International Journal of Molecular Sciences 21, no. 11 (May 28, 2020): 3827. http://dx.doi.org/10.3390/ijms21113827.
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Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.
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Xing, Jing-Jing, Jin-Gang Hou, Ying Liu, Ruo-Bing Zhang, Shuang Jiang, Shen Ren, Ying-Ping Wang, et al. "Supplementation of Saponins from Leaves of Panax quinquefolius Mitigates Cisplatin-Evoked Cardiotoxicity via Inhibiting Oxidative Stress-Associated Inflammation and Apoptosis in Mice." Antioxidants 8, no. 9 (September 1, 2019): 347. http://dx.doi.org/10.3390/antiox8090347.
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Background: Although kidney injury caused by cisplatin has attracted much attention, cisplatin-induced cardiotoxicity is elusive. Our previous studies have confirmed that saponins (ginsenosides) from Panax quinquefolius can effectively reduce acute renal injuries. Our current study aimed to identify the potential effects of saponins from leaves of P. quinquefolius (PQS) on cisplatin-evoked cardiotoxicity. Methods: Mice were intragastrically with PQS at the doses of 125 and 250 mg/kg daily for 15 days. The mice in cisplatin group and PQS + cisplatin groups received four times intraperitoneal injections of cisplatin (3 mg/kg) two days at a time from the 7th day, respectively. All mice were killed at 48 h following final cisplatin injection. Body weights, blood and organic samples were collected immediately. Results: Our results showed that cisplatin-challenged mice experienced a remarkable cardiac damage with obvious histopathological changes and elevation of lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB) and cardiac troponin T (cTnT) concentrations and viabilities in serum. Cisplatin also impaired antioxidative defense system in heart tissues manifested by a remarkable reduction in reduced glutathione (GSH) content and superoxide dismutase (SOD) activity, demonstrating the overproduction of reactive oxygen species (ROS) and oxidative stress. Interestingly, PQS (125 and 250 mg/kg) can attenuate cisplatin-evoked changes in the above-mentioned parameters. Additionally, PQS administration significantly alleviated the oxidation resulted from inflammatory responses and apoptosis in cardiac tissues via inhibition of overexpressions of TNF-α, IL-1β, Bax, and Bad as well as the caspase family members like caspase-3, and 8, respectively. Conclusion: Findings from our present research clearly indicated that PQS exerted significant effects on cisplatin-induced cardiotoxicity in part by inhibition of the NF-κB activity and regulation of PI3K/Akt/apoptosis mediated signaling pathways.
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Lindenkamp, Nicole, Katja Peplinski, Elena Volodina, Armin Ehrenreich, and Alexander Steinbüchel. "Impact of Multiple β-Ketothiolase Deletion Mutations in Ralstonia eutropha H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers." Applied and Environmental Microbiology 76, no. 16 (July 2, 2010): 5373–82. http://dx.doi.org/10.1128/aem.01058-10.
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ABSTRACT β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3′-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single- and multiple-deletion mutants were generated to investigate the influence of these β-ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16Δ2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16Δ2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth β-ketothiolase or a combination of several of the other β-ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.